Defective Ribonucleoproteins, Mistakes in RNA Processing, and

Subscriber access provided by NEW YORK UNIV

Current Topic/Perspective

Defective RNPs, Mistakes in RNA Processing and Diseases Mia K. Mihailovic, Angela Chen, Juan C. Gonzalez-Rivera, and Lydia M. Contreras Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b01134 • Publication Date (Web): 16 Feb 2017 Downloaded from http://pubs.acs.org on February 20, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

58x45mm (300 x 300 DPI)

ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. RNA processing within RNPs. RNAs and RBPs associate to form RNPs, complexes which facilitate a variety of mRNA processing steps, including splicing, stabilization, translocation, translation, sequestration and degradation. Upon transcription, hnRNPs prepare the pre-mRNA for splicing performed by snRNPs. In the nucleolus, RNA polymerase I and III, with the assistance of snoRNPs, ribosomal proteins and nonribosomal factors, transcribe rRNA precursors that assemble into functional ribosomes upon cytoplasmic translocation. Concomitantly with splicing, hnRNPs stabilize the transcript via 5’ capping and 3’ polyadenylation. Next, mRNP complexes guide the mature transcript into the cytoplasm, where the mRNA is translated or stored into SGs or P-bodies for inhibition or degradation, respectively. 179x139mm (300 x 300 DPI)


Page 2 of 56

Page 3 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 2. Defective RNP associations may be attributed to malfunctions in RNA-RBP crosstalk by various potential modifications. Altered RNP assembly and function can be caused by mutations or modifications to the RNA or RBP, or both. Such events change inherent structural or chemical composition and have the capacity to alter preferred binding partners, binding affinity, or enzymatic activity to modify and deregulate RNA-processing. 83x48mm (300 x 300 DPI)


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3. Disease-specific examples of defective RNA-processing steps. A. Defective splicing in AD. Upregulation of hnRNPA1 in AD patients causes alternative splicing of RAGE pre-mRNA resulting in a higher ratio of toxic mRAGE to non-toxic esRAGE isoform. mRAGE binds to the Aβ peptide, inducing an intracellular signaling cascade that triggers inflammation and subsequent cell death. B. Defective stabilization in FTD. In FTD, defective FUS likely lacks inhibitory control over deadenylase PAN2, leading to the destabilization and premature degradation of mRNAs such as GluA1. C. Defective translocation in SMA. SMN defects inhibit mRNP formation and subsequent β-actin mRNA axonal translocalization, preventing localized translation of β-actin. Such defective trafficking causes neurotransmitting defects due to decreased structural integrity. D. Defective translation in cancer. Upon high translational demand, 5S rRNPs assemble into matured preribosomal particles, abandoning their MDM2 target. Once free, MDM2 marks tumor suppressor p53 for degradation, inhibiting the desired apoptosis response in diseased cells. E. Defective aggregation in ALS. Hexanucleotide repeat expansions within the C9orf72 gene alter cellular function at both the RNA and protein level, to inhibit SG formation by sequestering necessary proteins such as TDP-43 and FUS and disturb nucleocytoplasmic transport by interacting with nucleolar and import proteins, respectively. 177x173mm (300 x 300 DPI)


Page 4 of 56

Page 5 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 4. Mechanisms, benefits, and drawbacks of RNA-based therapies used to treat RNA-processing defects within neurodegenerative disease and cancers. A. Synthetic siRNA and miRNA are commonly designed for mRNA inhibition and cleavage. Their silencing efficacies rely on processing by the endoribonuclease Dicer B. Antisense oligonucleotides do not require cellular processing to interact with mRNA targets to induce cleavage or alternative splicing but require stabilization to protect from ribonucleases. 152x83mm (300 x 300 DPI)


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 56

Defective RNPs, Mistakes in RNA Processing and Diseases Mia K. Mihailovic‡, Angela Chen‡, Juan C. Gonzalez-Rivera‡, Lydia M. Contreras* McKetta Department of Chemical Engineering, University of Texas at Austin, 200 E. Dean Keeton St., Stop C0400, Austin TX 78712

ABSTRACT Ribonucleoproteins (RNPs) are vital to many cellular events. To this end, many neurodegenerative diseases and cancers have been linked to RNP malfunction, particularly as this relates to defective processing of cellular RNA. The connection of RNPs and diseases has also propagated a shift of focus onto RNA-targeting from traditional protein-targeting treatments. However, therapeutic application development in this area has been limited by incomplete molecular insight of the specific contributions of RNPs to disease. This review outlines the role of several RNPs in diseases, focusing on molecular defects in processes that affect proper RNA handling in the cell. This work also reviews the contributions of recently-developed methods to understanding RNP association and function. We review progress in this area by focusing on molecular malfunctions of RNPs associated with the onset and progression of several neurodegenerative diseases and cancer and conclude with a brief discussion of RNA-based therapeutic efforts.

1 ACS Paragon Plus Environment

Page 7 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

INTRODUCTION Ribonucleoproteins (RNP) are complexes formed from the association of RNA-binding proteins (RBP) with an RNA. RNPs are critical for maintaining normal RNA metabolism by facilitating the vast majority of RNA processing steps. These post-transcriptional processing events include splicing, subcellular localization, translation, and degradation through a number of interactions that are often fairly conserved across eukaryotes.1,

2

Although the presence of

RNPs was recognized over half a century ago, only within the last few decades has significant attention been placed on the functional roles of RNPs in disease.3, 4 Beginning with the finding of potential defective assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs) in spinal muscular atrophy (SMA) by Liu et al.5, we have seen a recent paradigm shift in understanding disease onset and progression through the lens of RNA-processing malfunctions.5 To date, hundreds of RNPs have been identified and typically fall into a series of broad families based on the RNA-processing events that they facilitate, listed in Table 1. RNPs have been implicated in a wide range of diseases, demonstrating the ubiquity of posttranscriptional processing defects in human health.19, 20 For example, mutations in members of the hnRNP family of proteins, which are involved in splicing regulation and assist with nucleocytoplasmic shuttling, have been shown to promote cancer progression.21 Additionally, persistence of large mRNP complexes such as stress granules and P-bodies, which function as transcriptional and translational cellular regulators, has also been linked to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), or frontotemporal lobar degeneration (FTLD), and cancer.22 Due to the prevalence of deregulated splicing in neurodegenerative diseases, snRNPs, which make up the spliceosome, have been linked to the progression of ALS and Alzheimer’s disease (AD).23


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 56

Neurons are more vulnerable to cellular defects due to the fact that they are rarely replenished compared to other cell types. This longer lifespan therefore enables accumulation of deleterious mutations and provides a means for mutations to spread amongst a population of cells.20 Thus, we review specifically the role of RNPs in RNA-processing defects commonly seen in neurodegenerative diseases, such as ALS, SMA, and AD. Additionally, we discuss the role of RNPs in cancer as the uncontrolled proliferation of cells necessitates significant changes in normal RNA metabolism. This review also discusses how novel and improved biochemical methods are being applied for the generation of new mechanistic findings that allow better understanding of the role of various RNPs in diseases. A few in particular that we cover are single-molecule studies that offer insight to dynamic complex assembly, sophisticated prediction tools to assess viable RNA-RBP partners, mRNA interactome methods to elucidate characteristic RBP attributes of the RNPs, and the means to determine kinetic parameters such as binding and dissociation constants. Finally, we discuss recent advances in therapeutic development as specifically related to RNA-based therapies. NATIVE CELLULAR PROCESSING PATHWAYS FACILITATED BY RNPs AND THE ROLE OF RNPs IN STRESS RESPONSE From the synthesis by RNA polymerase II until degradation, RNAs undergo a myriad of life stages facilitated by RNPs. RNPs play critical roles in maintaining RNA homeostasis in normal and abnormal cellular conditions. For instance, in eukaryotes, RNPs coordinate cellular processes that span from splicing, stabilization, translocation, sequestration, and degradation, as seen in Figure 1. One of the first RNA processing events, co-occurring with transcription, is enhancement of pre-mRNA stability and protection via 5’ 7-methylguanylate capping by hnRNP formations containing RNA triphosphate, guanylyltransferase and methyltransferase.24,

25


Page 9 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Additionally, the 3’ end of the transcript is stabilized by hnRNPs such as the cleavage and polyadenylation specificity factor (CPSF) and poly(A) polymerase via assembly of a poly(A) tail of approximately 250 adenine residues. Due to the protective role of the poly(A) tail against ribonucleases,26 cytoplasmic modulation of this 3’ transcript stabilization has been associated with diseased phenotypes in ALS.27 Upon successful transcript stabilization, the spliceosome, which is composed of various snRNPs depending on spliceosome type (Table 1), is recruited to the transcript and performs alternative splicing through the removal of introns and rearrangement of exons.28, 29 As such, compositional or expression changes in snRNPs and hnRNPs have been implicated in numerous diseases, including SMA, ALS and AD.30 After splicing, the mRNA, in association with hnRNPs, is shuttled from the nucleus to the cytoplasm through the nuclear pore complex for translation. It is worthwhile to note that sequestration of transport-facilitating proteins, such as poly-A binding proteins, has also been linked to ALS phenotypes.31, 32 Once in the cytoplasm, mRNA can undergo translation within large ribosomal RNP complexes or associate into mRNP complexes known as P-bodies and SGs,33 which are dynamic higher-order RNP structures that offer tight control of the functional RNA pool in response to cellular needs or stress via translation inhibition or transcript degradation.33 Under stress, mRNAs can be sequestered in SGs consisting of translational factors and associated RBPs that disassemble once environmental circumstances no longer require mRNA sequestration to allow re-initiation of mRNA translation. As shown in Figure 1, the previouslysequestered mRNAs may also be transferred to P-bodies for further silencing or degradation, illustrating the dynamic exchange of mRNAs between the two complexes, ultimately depending on cellular demand.34 In contrast to SGs, P-bodies are void of translational factors, instead consisting of miRNAs and proteins involved with silencing facilitation, decapping, and


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 56

deadenlyation.34 Mutations in the prion-like domains of RBPs that facilitate RNA binding under stress conditions have been also associated with toxic hyperaggregates, as seen in ALS, FTD, AD, fragile X syndrome, Huntington’s disease (HD), etc.35, 36 Importantly, these aggregates can sequester proteins, mRNA, or other cellular factors necessary for proper cellular function. For example, HD-associated poly-glutamine-truncated Huntington gene (HTT) has been observed to sequester transcription factors such as general TATA box binding protein and co-transcriptional activators.36 MOLECULAR DEFECTS ON RNP COMPONENTS Disruptions to RNP function can be caused by single-point mutations or chemical modifications that manifest in either the RNA or protein component (Figure 2). These alterations affect multiple properties of RNP subunits such as coding capacity, molecular structure, binding regions, catalytic activity and respective post-transcriptional or post-translational processing capabilities that eventually manifest as pathologies. As seen in Figure 2, the inherent crosstalk between the RNA and protein components of a RNP exacerbates the effect of a mutation due to the required dynamic assembly and interaction of RNA and RBPs. Thus, this section explores molecular changes in the RNP subunits that can alter proper RNP assembly and function, ultimately leading to particular diseases. Natural pathways known to edit RNA sequences via RNA modifications for functional regulation in eukaryotes includes (i) the conversion of adenosine to inosine (A-to-I), (ii) the conversion of cytidine to uridine, and (iii) 2’-O ribose methylations.37 Deregulation of such edits has numerous effects on the resulting RNAs as they can alter coding capacity, target binding affinity and processibility. For example, inosine preferentially base pairs with cytosine, and therefore is interpreted as guanosine during RNA hybridization. As such, A-to-I editing of the


Page 11 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

pre-mRNA of the G protein-coupled receptor, 5-HT2C, modifies its transient interactions with snRNPs to induce alternative splicing events linked with Prader-Willi syndrome.37 On the other hand, deviations from normal physiological conditions can lead to alteration of sequence-defined RNA characteristics via unintended mutations and chemical modifications. Point mutations have been shown to influence RNA secondary structure and associated enzymatic activity. For example, in patients with hematologic diseases such as dyskeratosis congenita (DKC) and aplastic anemia, gene mutations in hTERC, the telomere template RNA, are believed to disrupt a structured helix and thus render the RNA “unreadable” by telomerase.38 In contrast, stressinduced modifications, such as methylations and oxidations, can alter binding affinity or facilitate non-canonical base-pairing, resulting in altered RNA function.39,40 Modified nucleobases have been shown to disrupt translational processing by modifying the functional interaction between the mRNA and the ribosome. This abnormal processing of oxidized mRNAs leads to modified protein products and short peptides that contribute to the accumulation of protein aggregates as observed in AD.41 More recently, an 8-oxo-7,8-dihydoguanosine (8-oxoG) modification in the miRNA, miR-184, was shown to induce alternative target silencing and subsequent apoptosis in rat heart cells.42 This observation is intriguing, as it may lead to new research linking oxidations within regulatory RNAs to pathologies. RNP components can also suffer aberrant modifications at the protein level that alter the biogenesis and homeostasis of RNPs. Variations in the amino acid sequence of RBPs can ultimately (i) alter their subcellular localization, (ii) modify their binding affinity for RNA targets, (iii) disrupt their association with protein partners, (iv) deregulate their normal biochemical activity, and (v) induce protein aggregates. The proper subcellular localization of protein components is essential in RNP biogenesis as it ensures the correct interaction and


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 56

assembly with the target RNA ligand and subunits. For instance, autosomal recessive mutations that cause mislocalization of TCAB1, a Cajal body RNA chaperone that recruits the telomerase protein component (TERT), lead to DKC and oncogenic genome instability.43 Molecular defects in RNA-binding motifs can additionally reduce affinity for their RNA targets. For example, missense mutations in the KH-type RNA-binding domain in the fragile X mental retardation protein (FMRP) inhibits RNA binding, resulting in behavior abnormalities characteristic of Fragile X Syndrome, a genetic cause of autism and mental retardation.44 Similarly, point mutations found in the RNA-recognition motif (RRM) of the RBP nuclear poly(A)-binding (PABPN1) cause a decrease in its affinity for RNA and subsequently deregulate polyadenylation by inhibiting interaction between RNA and poly-A polymerase, leading to the onset of oculopharyngeal muscular dystrophy.45 Mutated proteins can alternatively perturb assembly of RNP precursor complexes, alter enzymatic activity, or induce aggregations. For example, in patients diagnosed with SMA, missense mutations in the Tudor domain of the survival motor neuron (SMN) protein reduce its ability to associate with Sm proteins and protein subunits in U snRNPs, resulting in deficient U snRNP biogenesis.46 Additionally, mutations in the catalytic RT domain of TERT suppress telomerase activity but not RNA binding, reducing catalytically active telomerase which can lead to oncogenesis.47 Moreover, genomic mutations on the hnRNP fused in sarcoma (FUS), also known as translocated in liposarcoma (TLS), TAR DNA-binding protein 43 (TDP-43), and the ubiquitin-binding protein p62 enable colocalization of these proteins in neuronal cytoplasmic inclusions.11 These mutations were identified in ALS patients, evidencing the classical association of abnormal protein aggregates with neurodegenerative pathologies. Finally, protein level chemical modifications can deregulate the biogenesis and function of RNPs, as shown by unintended demethylations in the arginine residues of PABPN1. These


Page 13 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

demethylations are associated with muscular dystrophy and can induce the release of the RNA ligand by favoring its interaction with the import receptor transportin compared to PABPN1.48 Likewise, hypo-methylation in arginine residues in FUS causes overly tight protein-protein interaction with transportin, driving FUS into the cytosol while the nucleus becomes devoid of FUS RNPs, an event linked to ALS and FTD.49 ROLES OF DEFECTIVE RNA-PROCESSING STEPS IN DISEASE In neurodegenerative diseases and cancer, multiple RNA-processing steps can be found defective on the molecular level. Here we choose a representative set of diseases— SMA, ALS/FTD, AD, and cancer— and a subset of associated RNPs to illustrate various defective RNA-processing steps in disease, whose similarities and differences may offer insight into approaching the study of RNPs involved in other neurodegenerative diseases. SMA is a mechanistically well-characterized neurodegenerative disease associated with degradation of SMN and skeletal atrophy.

13

Functional SMN has many roles, including mRNA transport,

editing, and splicing; however, one of the better understood behaviors of SMN is its facilitation of the biogenesis of snRNPs.50 Specifically, SMN, in complex with a multitude of Gemin proteins, loads Sm protein onto nascent snRNAs upon their export to the cytoplasm, creating snRNPs that form spliceosomes upon their return to the nucleus.29, 51, 52 ALS and FTD are similarly well-characterized diseases whose side-effects include lower and upper motor neuron degeneration, causing muscular atrophy in ALS and frontal/temporal cortex neuronal atrophy linked to alterations in personality and behavior in FTD.10 While these pathologies are inherently unique, some cases of ALS also display FTD-characteristic symptoms and pathogenic mechanisms, and vice versa, prompting the consideration of these neurodegenerative diseases on a continuum.10 Notable proteins associated with this continuum


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 56

are ubiquitous hnRNPs TDP-43 and FUS/TLS.53 These hnRNPs have considerable functional and structural homology; for example, both are involved in transcriptional initiation and repression, RNA stabilization and splicing facilitation, and share similar features such as the RRM domain and a C-terminal glycine-rich region in which most ALS-associated mutations occur.54, 55 More recently, attention has been given to understanding the role of RNA-processing defects in the onset and progression of AD and cancer. Although the cause-effect relationship of RNP defects is not well understood, numerous studies have shown that AD-implicated hnRNPs A1 and K, among others, have similar activity to oncogenic proteins and play significant roles in several cancers.8 Each of the aforementioned disease phenotypes exhibit multiple defective RNA-processing steps, many of which are still not understood as a direct cause or consequence of the disease. Here we elucidate some examples of disease-specific defective RNA-processing events (Figure 3) to compare and contrast RNP –affected mechanisms between diseases. Splicing malfunctions implicated in disease Splicing defects are a hallmark of many neurodegenerative diseases and cancer, as downstream consequences directly affect the functionality of the cellular protein pool. Causes of splicing malfunctions can be correlated to different complexes that facilitate splicing directly, such as snRNPs that form the spliceosome, or indirectly, through hnRNPs that facilitate splice-site recognition. Interestingly, hnRNP inability to regulate splicing inhibition is common to both AD and ALS, while incorrectly-processed snRNPs cause SMA phenotypes. In SMA, splicing alterations linked to SMN mutants are a well-established pathogenic mechanism supported by (i) reported deficient levels of snRNPs in SMA, (ii) a correlation


Page 15 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

between snRNP levels and disease progression, and (iii) relieved ALS pathogenicity upon addition of snRNPs.13, 24 SMA fatality risk is highest for infants, a phenomenon that can possibly be explained by the observation that snRNP biogenesis is greatest during the first two weeks post-partum in mice.56 SMN mutations have been reported to preferentially decrease expression levels of snRNPs comprising the minor spliceosome, which is estimated to regulate splicing for roughly 1% of eukaryotic genes.13,

57, 58

Specifically, SMN1 interference in mouse embryonic

fibroblasts has been shown to cause irregular maturation and alternative splicing of U12-intronic mRNAs, resulting in decreased expression levels. Interestingly, knockdown of stasimon, a U12intron containing gene, in Drosophilia induced a similar phenotype to that observed in SMA. 13 In contrast to SMN, malfunctioning splicing mechanisms in AD and ALS have been correlated to defects in hnRNPs that function as molecular splicing facilitators. These RNPs are known to facilitate a variety of nuclear RNA processing steps by maintaining pre-mRNA structure.59 In AD, the receptor for advanced glycation end products (RAGE) has two major alternative splicing isoforms that play crucial roles: the full-length membrane bound isoform (mRAGE) and the Cterminally truncated secretory RAGE (esRAGE).60 mRAGE is a receptor for the amyloid-β (Aβ) peptide and was found to have increased expression in the brains of AD patients while esRAGE functions as a decoy receptor and mitigates the activation of mRAGE pathways by removing or neutralizing mRAGE ligands.38, 61, 62 Overexpression and knockdown of hnRNP A1, a splicing repressor that promotes distal splice site selection,21 correlated with an increase and decrease in the ratio of mRAGE to esRAGE, respectively (Figure 3A). Furthermore, hnRNP A1 protein and mRNA levels are completely absent in entorhinal cortexes of AD patients. Additionally, it has been shown that hnRNP A1 overexpression induces alternative splicing of the amyloid precursor protein (APP) mRNA to reduce Aβ levels;63 these findings suggest that hnRNP A1 plays an


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 56

important role in regulating Aβ by modulating APP, in addition to mRAGE mRNA and protein.64 Studies have also attributed changes in ALS-associated aberrant alternative splicing to the RBP TDP-43.65 Interestingly, numerous studies have recently suggested the regulation of TDP-43 splicing factor by other ubiquitously-expressed hnRNPs such as hnRNP U and hnRNP A2. This observation is supported by (i) co-immunoprecipitation experiments in Drosophila

66

,

(ii) hnRNP influence on TDP-43-dependent exon 17b splicing in sortilin 1 neurotrophic receptor (SORT1)

67

and (iii) reversed TDP-43-induced pathological progression with hnRNP

overexpression in motor neuronal cells. 9 This may imply that hnRNPs, which normally mediate TDP-43 splicing, lack control over TDP-43 mutants in ALS phenotypes. Further investigation of hnRNP molecular associations may be a promising strategy to identifying mechanistic defects in neurodegenerative diseases exhibiting altered splicing patterns. Stabilization malfunctions implicated in disease In addition to RNA processing malfunctions in splicing, alterations in RNA stabilization facilitated by polyadenylation have also been implicated in disease progression. For instance, FUS mutations may deplete mRNA levels, including GluA1, to induce characteristic FTD behaviors. Under normal conditions, FUS is clustered around an alternative polyadenylation site in neuronal cells, utilizing its relative position to regulate the poly(A) tail length to control mRNA stability and subsequently protein levels.68 Specifically, it was hypothesized that FUS sequesters the deadenylase catalytic subunit, PAN2, on PABPC1 (poly(A) binding protein cytoplasmic 1) and prevents it from attacking the poly(A) tail, enabling improved mature mRNA stability in the cytoplasm. 27 In the absence of FUS, GluA1 is destabilized and degraded, altering the functional protein pool to induce FTD-like deficient synaptic function (Figure 3B). Other studies have shown severe deregulation of polyadenylation events in diseases such as the RNA-


Page 17 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

mediated myotonic dystrophy.

69

These results suggest that many of these malfunctions are

facilitated by sequestration of crucial polyadenylation regulators, with loss of these regulators resulting in aberrant mRNA stability and translocation. Translocation malfunctions implicated in disease The inability of mRNAs and proteins to be transported to their proper locations can have widespread effects on dependent downstream processes. Defective translocation across the nuclear membrane has been implicated in both ALS and AD. ALS and AD-associated translocation defects are observed to inhibit cytoplasmic and nuclear translocation, respectively. For example, C9orf72 RNA containing hexanucleotide repeat expansions (HREs), associated with ALS, has been observed to form nuclear inclusions in association with trafficking proteins.70,71 Additionally, overexpression of HRE-containing C9orf72 in neurons induced an accumulation of mRNAs in the nucleus, accompanied by nuclear PABPc inclusions.72 This suggests that cytoplasmic translocation in ALS is deregulated on multiple levels: (i) trafficking proteins are physically unavailable due to sequestration and (ii) mRNA transcripts are unstable and thus unqualified for translocation.72 In AD, defective nuclear translocation of U1 snRNP, one of the five snRNPs that constitute the major spliceosome, results in a loss of function for the spliceosome and subsequent aggregation and subcellular mislocalization of proteins and RNA. Specifically, studies during early disease development of brain-insoluble proteins found widespread accumulation of extranuclearaggregated U1 snRNP in neuronal bodies accompanied by the presence of U1-specific proteins U1-70K and U1A in neurofibrillary tangles.73 These studies lend additional credence to the possibility of misregulated translocation due to sequestration of key translocation-facilitating proteins.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 56

In contrast to ALS and AD, nucleocytoplasmic transport in cancer is highly efficient and is, in fact, targeted for inhibition to slow cancer progression. hnRNP A1 is known to be heavily involved in the nucleocytoplasmic shuttling and splicing of many mRNA transcripts, including known oncogenic proteins. Indeed, studies have shown direct correlation of hnRNP A1 expression with protumorgenic embryonic M2 isozyme of pyruvate kinase (PK-M2) and cancer progression levels.74,

75

Taking advantage of this, reports have demonstrated how chemically-

induced inhibition of hnRNP A1 nuclear transport can be used for treatment of prostate cancer by promoting apoptosis through the cytoplasmic accumulation of hnRNP A1 and increased recruitment of SGs.69, 76, 77 Additionally, expression of a shuttling-deficient mutant suppressed tumorigenesis in bone marrow cells overexpressing oncoproteins, further demonstrating cellular dependence on nucleocytoplasmic transport for maturation and proliferation.78 It is interesting to note that malfunctions in normal translocation activity promote progression of ALS and AD, but actually block the progression of cancer, which most frequently targets non-neuronal cells. Abnormal translocation is also observed in SMA phenotypes, specifically in relation to SMNfacilitated axonal trafficking. Under normal cellular conditions, SMN interacts with various RBPs involved in neuronal mRNA trafficking to facilitate association with motor proteins that travel between the synapse and dendrites. 79, 80 However, in SMA, axonal trafficking is defective and causes inadequate local translation, often compromising cellular structural integrity. For example, SMN defects cause decreased expression of KH-type splicing regulatory protein (KSRP) and β-actin mRNA-binding protein IMP1, resulting in limited axonal translocation of βactin mRNA, which is important for proper axon branching (Figure 3C).16, 17, 79, 81 More recently, a genome-wide approach identified over 200 mRNAs that colocalize with SMN in motor neuron cells, suggesting that the pool of SMA-afflicted mRNAs is much larger than expected.82 As the


Page 19 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

unique morphology and transmitting in neurons requires uncharacteristically far translocations compared to other cell types, neuronal translocation may be particularly sensitive to SMN mutations.83 Such considerations offer a mechanistic justification of characteristic degradation specificity towards neurons and muscles seen in SMA and other neurodegenerative diseases. Translation malfunctions implicated in disease ALS, FTD and cancer are all characterized by a series of disorders that lead to adverse alterations in the assembly and function of ribosomes. Although through unique means, these diseases are all characterized by production of undesired proteins. Additionally, various proteins act to deregulate translation initiator factors in a stress response-like mechanism in AD, SMN and cancer. Interestingly, many of the identified mechanisms function as feed forward loops, where specific proteins or RNAs regulate activation or expression of an effector molecule by forming transient RNPs, stimulating the activity of the effector and leading to sustained pathological defects. For instance, defective and non-canonical translation in ALS and FTD is triggered by HREs in the C9orf72 gene. Mutant C9orf72 proteins lead to disease pathology by loss of their function, accompanied by a gain of function of C9orf72-associated mechanisms driven by the RNA species encoded in the HRE.84 At the RNA level, mutation-altered C9orf72 function includes (i) aberrant aggregation that sequesters key RNA-binding proteins such as hnRNP TDP-43 in the nucleus and (ii) formation of diverse stable RNA secondary structures like G-quadruplexes by the HRE that impair ribosomal elongation and induce ribosome frameshifting.84 Formation of RNA G-quadruplexes increases non-canonical RANT-dependent translation of the HRE, in which dipeptide repeat expression is allowed in all three reading frames without an AUG start


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 56

codon.85 Simultaneously, these G-quadruplexes prevent ribosomal association with non-mutated C9orf72 to further support a circuitry propagating abnormal C9orf72 protein products. 84 On the other hand, in cancer, abnormal stimulation of cell proliferation requires an increase in protein synthesis, which is accomplished by upregulation of ribosome biogenesis. This perturbation in the rate of ribosome assembly triggers a p53-dependent checkpoint pathway that attempts to directly reduce the ribosome biogenesis and cell growth.86 However, this desired regulation of p53 is inhibited by the action of E3 ubiquitin-protein ligase (MDM2). In response to the increased demand of translation, ribosomal proteins bound to MDM2 are recruited for ribosome assembly releasing MDM2. MDM2 is then available to control p53 activity by either binding to the protein and interfering with its function, or by recruiting a E3 ubiquitin ligase to promote its degradation by the proteasome (Figure 3D).86 Another RNP-associated mechanism by which diseased cells control translation is by acting on translation initiation factors and modulating their phosphorylation levels. Interestingly, these mechanisms take advantage of stress-induced regulators that invoke molecular checkpoints to control translation. For instance, loss of SMN function in SMA is known to reduce phosphorylation of the eukaryotic initiation factor eIF2α, whose phosphorylated form acts as an environmental stress-responsive regulatory element. Phosphorylation of eIF2α prevents the recruitment of the initiator methionyl-tRNA (Met-tRNAiMet) to the ribosome and inhibits translation activation.50 Similarly, phosphorylation of the eukaryotic initiation factor eIF4E is implicated in AD.87 This factor plays a key role in regulation of the microtubule-associated Tau protein mRNA, as abnormally phosphorylated Tau is the major protein component of neurofibrillary tangles present in AD pathology.87 In cancer, the RBP HUR forms transient mRNPs to modulate the stability and translation of multiple transcripts encoding effectors of


Page 21 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

tumorgenesis such as the tumor suppressor p53, oncogenes (c-fos, c-Myc), growth factors (VEGF, TGFβ, TNFα) and apoptosis-related factors (Bcl-2, Mcl-1).88 Additionally,

HUR

interacts at the RNA and protein level with eIF4E to specifically increase the expression of oncogenic mRNAs by enhancing stability of the eIF4E mRNA, which stimulates translation of other pro-oncogenic targets and invokes a continued tumorgenesis process. 88 Aggregation and stress granule biogenesis malfunctions implicated in disease In ALS, mutant C9orf72 causes disorders in protein aggregation through the actions of its RNA and aberrant protein species encoded in the genomic nucleotide repeat expansion. At the RNA level, HRE-containing C9orf72 transcripts accumulate as nuclear RNA foci, whose formation is implicated in the sequestration of numerous RBPs and subsequent alteration of RNPs activity.

84

For example, mutant C9orf72 RNA sequesters hnRNPs TDP-43 and FUS into

aggregates, silencing their specific activity in the biogenesis of SGs.7 Indeed, specific RNPassociated RNA and RBPs interfere with SG biogenesis by sequestering essential proteins and by modulating levels of initiation factors that activate SG biogenesis (Figure 3E). Furthermore, HRE-containing C9orf72 strongly interacts with hnRNP H, decreasing global splicing in motor cortex and cerebellum tissues.7 C9orf72-mediated sequestration also influences the assembly of adenosine deaminase RNA-specific B2 (ADARB2) mRNP, and hnRNP A1, hnRNP A2/B1 and hnRNP A37. Translation of mutant C9orf72 mRNAs produces dipeptide repeat protein products that are highly prone to aggregation and are known to impair nucleocytoplasmic trafficking by interfering with the dynamics of nuclear pore complexes, import factors and nucleolar proteins (Figure 3E).84,89 In SMA, deregulation of SMN protein has been shown to similarly reduce the cellular capability to generate SGs in response to stresses.

50

SMN has been linked to the biogenesis of


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 56

SGs by directly interacting with associated assembly proteins. For example, SMN colocalizes with TIA-1/R and the G3BP, two protein assemblers of SGs, suggesting a potential pathway for SMN-mediated SG biogenesis.50 Additionally, functional SMN modulates the phosphorylation of the initiation factor eIF2α, a known promoter of SG formation. 50 In AD, Aβ presence propagates aggregation on two levels, by (i) decreasing the solubility of TDP-43 and Tau and (ii) inducing plaque inflammation that signals further Aβ synthesis.90 Specifically, decreased solubility of TDP-43 and Tau facilitates their deposition and coexistence in neuronal cytoplasmic inclusions and dystrophic neurites.90 Furthermore, Aβ accumulation in plaques incites a deregulated inflammation response allowing the release of pro-inflammatory cytokines such as tumor necrosis factor (TNF-α), interleukin-6 (IL-6), or IL-1β.

90

Interestingly,

TDP-43 is upregulated in response to such neuroinflammation, enabling a cycle in which local inflammation events caused by the presence of Aβ induce accumulation of TDP-43 and sustain pathological aggregates. 90 NOVEL

METHODS

SUPPORTING

ADDITIONAL

MECHANISTIC

AND

FUNCTIONAL RNP INSIGHTS Early studies on RNPs utilized a combination of traditional genetics and biochemical techniques such as affinity chromatography, protein precipitation, immunoprecipitation, and electrophoresis to identify, purify, and characterize the role of these RNA-protein complexes in eukaryotic cellular processes.

91-93

As interest in RNPs has increased over the years due to their

involvement in numerous human diseases, the need for greater mechanistic insight into their roles in RNA processing and disease progression is apparent.

In

recent

years,

immunoprecipitation based sequencing approaches such as RIP-seq and CLIP-seq, RT-PCR, and RNA silencing by siRNAs have been the workhorses of studies in SMA, ALS, AD and


Page 23 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

numerous cancers. 94, 95 Typically, these experiments have consisted of immunoprecipitation of a protein or RNA of interest or overexpression/knockdown of a gene. Recently, the advent of highthroughput sequencing has been used to identify interaction partners or to analyze the phenotypical changes respectively. Importantly, use of these techniques has helped elucidate the functional role of RNPs such as FUS, TDP-43, U1 snRNP, and more, in abnormal RNAprocessing steps by identifying binding motifs and interaction partners in RNPs.

96, 97 98-100

We

will focus our discussion on a few specific approaches that have recently been developed to address limitations of previous methods and their applications in studying ALS, AD, SMA, and cancer. RNA-centric interactome methods show unique RBP partners and enriched RBP motifs associated with disease RNA-centric methods are being increasingly used to identify protein components of RNPs implicated in various diseases. Low-throughput methods such as biotinylated RNA-pulldown assays coupled to mass spectrometry (MS) analysis have offered valuable information to the interactome of ALS-implicated aberrant HRE occurring in the C9orf72 gene. Specifically, these methods have led to the detection of novel interactions with proteins involved in ribosomal function and RNA post-transcriptional modification, in addition to expected splicing proteins.101 However, high-throughput versions of these RNA pulldowns coupled to MS methods have offered even greater insight by enabling identification of unique RBP features and why these characteristics may be targeted in neurodegenerative disease.

100

One such high-throughput

method, termed RNA interactome capture, relies on in vivo covalent UV crosslinking of RNA with respective RBPs and poly(A) tail-dependent oligo(dT) capture to identify RBPs in complex with all mRNAs in the transcriptome.102 Conducting interactome capture facilitated identification


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 56

of 860 novel RBPs in HeLa cells, discovering many intrinsically disordered RBPs which were rich in short repetitive amino acid motifs. This discovery helped elucidate the mysterious RNP aggregation tendency that is characteristic of numerous diseases.102, 103 RNA Bind-N-Seq (RBNS) highlights structure and sequence specificity that govern RNA-RBP interaction In addition to identification of the proteins and RNAs that make up RNPs, it is also important to understand how key parameters such as the structure and sequence of the protein and RNA affect binding kinetics, which provide insights to disease mechanisms and aid therapeutic design. Several recent experimental methods have been introduced that allow users to study how RNA structure and sequence affect RBP binding and estimate kinetic constants. One such technique is RNA Bind-N-Seq (RBNS), which is a high-throughput in vitro method that can provide quantitative data on RNA binding events.

104

As an example, Lambert et al. successfully used

RBNS to identify known and secondary motifs of RBFOX2, MBNL1, and CELF1 proteins, obtain estimates of dissociation constants, and elucidate the impact of RNA structure on RBP binding.

104

Although there are numerous methods to identify binding motifs, few have the

ability to provide estimates of binding affinity, which is crucial knowledge for the development of therapeutic inhibitors or competitors. RBNS in tandem with CLIP-seq was also recently used to study three ALS-associated proteins, FUS, TDP-43, and TAF15. This study found that TAF15 binds to the GGUA motif and exhibited similar intronic binding patterns to FUS despite having little role in alternative splicing.97 These results support the hypothesis that neurodegenerative diseases like ALS are multilayer diseases occurring in several cellular processes. RBNS was also used to study RNA networks modulated by hnRNPA1B2, which has been linked to ALS and AD pathologies, identifying the UAGG motif in RNA as the binding motif of these hnRNPs.105


Page 25 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

These studies confirm that the use of RBNS facilitates high-throughput identification of binding motifs and underlying features of critical RNPs in disease. Predictive Software CatRAPID Can Inform Experimental Design to Elucidate Interactions Involved in Disease Mechanisms Functional domain characterization remains a considerable challenge due to the non-specificity of protein motif-based recognition techniques, as proteins recognize RNA by sequence, structure, or both.

103

The aforementioned RNA-interactome studies that support large-scale identification

of non-specific RBPs offer valuable, although low-resolution106, insights to functional protein domains without performing expensive, labor-intensive imaging experiments such as nuclear magnetic resonance spectroscopy or X-ray crystallography.106,107 Similarly, protein-centric studies like RNA Bind-N-Seq (RBNS) provide insight on RNA characteristics such as secondary structure and binding motifs that are favorable for promoting interaction with RBPs.108 These motif-based insights are valuable inputs for predictive programs that identify potential RNA or protein binding regions, likely RNA-RBP binding partners, and even three-dimensional RNPcomplex structure, through a process known as docking.103,106,107 CatRAPID-omics is a sequence-based algorithm that uses predictions of secondary structure, hydrogen bonding and van der Waals’ forces paired with a recognition domain search to identify likely binding partners and detect and rank their binding regions from a given transcriptome or proteome.109 Comparative analysis showed CatRAPID-omics could potentially predict a considerable portion of experimentally-verified ncRNA binding partners of AD and ALS-implicated hnRNP TDP43.110 Additionally, the program correctly identified a functional region within TDP-43 involved in autoregulation, which has been implicated in self-splicing-regulated propagation in ALS.111 Such prediction programs are informative to experimental design to reveal disease mechanisms.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 56

Single molecule techniques SiMPull and its extensions provide insights to the dynamic nature of RNA-RBP interactions Although advances in computational and experimental efforts towards elucidating the RNARBP interaction network and its effect on the role of RNPs in disease have been made, many of these methods are static and capture interactions only at a specific time. Thus, as a compliment to these methods, single molecule techniques provide the opportunity to visualize the dynamic behavior of RNPs and their binding partners. Single molecule pulldown (SiMPull) and its derivatives SNAP-SiMPull and SiMPull-FRET, fuse conventional pulldown assays with single molecule fluorescence microscopy or single molecule FRET to allow direct visualization of individual protein complexes. 112-114 Using these methods, studies have demonstrated the ability to detect single molecules of U1 snRNP and to investigate the dynamic nature of mammalian target of rapamycin (mTOR)-containing complex (mTORC) dimerization and the selectivity of rapamycin on mTORC stoichiometry.

114,115

The

ability to understand the dynamics of mTORC1 and mTORC2 is promising as mTOR is a regulatory protein associated with many cellular pathways like mRNA translation, ribosome biogenesis, autophagy, and metabolism. Additionally, mTOR deregulation has been linked to several types of cancer.

116

SiMPull-FRET was successful utilized to investigate pre-mRNA

splicing dynamics and revealed that the spliceosome achieves an accurate initial splice through a biased Brownian ratcheting mechanism.113,117 Since deregulated alternative splicing has been identified in ALS, SMA, AD, and even cancer, the capability to monitor spliceosome dynamics is a strong first step towards understanding the beginning of splicing defects and their proliferation through the cell.


Page 27 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

THERAPEUTIC EFFORTS TO CONTROL DEFECTIVE RNA-PROCESSING IN DISEASE Insights into the pathogenic mechanisms of neurodegenerative diseases and cancer have uncovered potential routes for successful therapeutic intervention. Some treatment approaches include replacing mutated genes by genetic therapy118,119, regenerating functional cells using stem cell technology120 or targeting molecular biomarkers via protein-based therapy121, small molecule inhibitors122 or RNA-interference (RNAi).123 However, targeting with synthetic RNA molecules has flourished due to its relatively easy synthesis and competitive cost compared to other approaches. This feed-forward strategy interferes with undesired or defective RNAs to prevent dysfunction of downstream cellular systems linked with pathological processes. As such, RNA-interference (RNAi) by small interfering RNA (siRNA) or microRNA (miRNA) are potential therapeutic methods for disorders associated with RNPs (Figure 4A). The therapeutic potential of these methods is promising due to the capability of targeting any desired transcript, overcoming one of the main limitations of the traditional small molecule therapeutics.123 This approach has been successfully applied for the treatment of cancer by using neutral liposomal particles to deliver siRNA targeting the mRNA of tyrosine kinase receptor EphA2, a receptor overexpressed in multiple cancers.124 Currently in clinical trials, this siRNA interferes with the mRNP machinery that facilitates EphA2 mRNA processing, subsequently triggering its turnover. RNAi therapeutics have also been effective in the treatment of a broad variety of pathologies by acting on mRNA 5’ and 3’ untranslated regions to deregulate interaction with RNA-binding proteins and interfere with mRNP dynamics. Some of these diseases include age-related macular degeneration, transthyretin amyloidosis, Hepatitis B and C, fibrosis and hypercholesterolaemia among others.125, 126


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 56

Another strategy for RNA silencing utilizes antisense oligonucleotides, which interact with their target RNA via perfect complementarity and block access to regulatory RNA sites through steric hindrance (Figure 4B). This strategy is often utilized due to its higher specificity and lack of dependence on enzymatic processing compared to RNAi counterparts. Oligonucleotide-based therapeutics are currently being tested in clinical trials for a variety of conditions such as cardiovascular diseases, metabolic and endocrine diseases, inflammatory diseases, ocular diseases and infectious diseases with three drugs already receiving approval by the US Food and Drug Administration (FDA) .127-130 One of these drugs is a splice-switching oligonucleotide (SSO) that relies on targeting pre-mRNAs to inhibit or stimulate exon inclusion, forcing snRNPs to consider alternative splicing sites. Multiple SSOs, such as Nusinersin, have been designed to mitigate SMA by interfering with intronic splicing silencer elements to enhance inclusion of exon 7 in SMN2 and increase production of full length SMN.129,131 Similarly, in AD, antisense oligomers have been used to enhance mRNP processibility by stimulating the inclusion of exon 19 in ApoER2, an apolipoprotein E receptor involved in learning and memory that has been showed to be downregulated in AD patients. 132 In cancer, antisense oligomers promote skipping of exon 6 and decrease abundance of the MSM4 oncogene, inhibiting melanoma cell growth.133 A second mechanism of antisense RNAs, known as gapmers, promotes an mRNP complex that cleaves the target RNA by endogenous RNase H.127 Gapmers have been utilized in ALS treatment to target mutants of the Cu/Zn superoxide dismutase, SOD1, that cause disease by a toxic gain of function in nearly 20% of familial ALS patients.134 Similarly, the gapmer ZD9150 promotes inhibition of the cancer-associated transcription factor STAT3 and is currently in clinical trials for cancer treatment.43 Additionally, numerous gapmers are in clinical trials to


Page 29 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

mitigate other diseases such as thrombosis, type 2 diabetes, obesity, HD, and asthma, among others.127 While therapeutic efforts are promising, there remain challenges, such as low in vivo efficacy and off-target effects, in their design and delivery to the target cells. Thus, it is important to consider the secondary structure of both the RNAi and the target RNA as low local accessibility of the RNAi recognition site weakens the overall thermodynamic stability of the duplex, compromising therapeutic efficacy.135 Additionally, optimizing RNAi folding can reduce nonspecific interactions with unintended transcripts. To achieve this aim, a multifactor approach is necessary, as single-feature analysis cannot fully explain the functional efficacy of therapeutic RNAi.135 Features such as GC content, duplex binding, RNAi folding, target site folding, and 5’end base have been considered in numerous pipelines for successful design of functional siRNAs.136 Therapeutic efforts, while still in development, have made significant progress with numerous treatments reaching late-stage development in cancer and neurodegenerative diseases. Although there has been tremendous success in the field, opportunities for improvement remain. Further development and commercial application of silencing technology should be possible through improvement of RNAi design and development of innovative delivery methods. Finally, improved accuracy and effectiveness of treatment depends largely on the continued study of RNA-processing mechanisms associated with diseases using novel, tailored approaches. TABLES Table 1. Categories of ribonucleoproteins (RNPs). Type of RNP Heterogeneous nuclear

Cellular activities Alternative splicing/

Examples hnRNP A16, hnRNP A37, hnRNP


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 56

transcriptional

K8, hnRNP A2B1, hnRNP U9,

regulation

TDP-4310, FUS/TLS11

Small nuclear

Components of the

Spliceosome subunits: U1, U2, U4,

ribonucleoprotein (snRNP)

spliceosome

U4, U5 snRNP (major)12, U11,

ribonucleoproteins (hnRNP)

U12, U4atac, U6atac, U5 (minor)13 Small nucleolar

Post-transcriptional

C/D box snoRNP and H/ACA

ribonucleoprotein (snoRNP)

modification of

snoRNP 14 ; U3 snoRNP 15

ribosomal RNA (rRNA). Pre-rRNA folding and processing. microRNA ribonucleoprotein

Gene-silencing

(miRNP)

RNA-induced silencing complex (RISC)

Messenger ribonucleoprotein

mRNA processing:

RNA-binding proteins associating

(mRNP)

transport, translation

with mature mRNA: e.g.,

and decay

IMP1/ZMP116, KSRP17 etc. Cytoplasmic mRNP granules: processing body (P-body), and stress granules (SG) 18

FIGURES


Page 31 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 1. RNA processing within RNPs. RNAs and RBPs associate to form RNPs, complexes which facilitate a variety of mRNA processing steps, including splicing, stabilization, translocation, translation, sequestration and degradation. Upon transcription, hnRNPs prepare the pre-mRNA for splicing performed by snRNPs. Concomitantly with splicing, hnRNPs stabilize the transcript via 5’ capping and 3’ polyadenylation. Next, mRNP complexes guide the mature transcript into the cytoplasm, where the mRNA is translated or stored into SGs or P-bodies for inhibition or degradation, respectively.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 56

Figure 2. Defective RNP associations may be attributed to malfunctions in RNA-RBP cross-talk by various potential modifications. Altered RNP assembly and function can be caused by mutations or modifications to the RNA or RBP, or both. Such events change inherent structural or chemical composition and have the capacity to alter preferred binding partners, binding affinity, or enzymatic activity to modify and deregulate RNA-processing.


Page 33 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 3. Disease-specific examples of defective RNA-processing steps. A. Defective splicing in AD. Upregulation of hnRNPA1 in AD patients causes alternative splicing of RAGE premRNA resulting in a higher ratio of toxic mRAGE to non-toxic esRAGE isoform. mRAGE binds to the Aβ peptide, inducing an intracellular signaling cascade that triggers inflammation and subsequent cell death. B. Defective stabilization in FTD. In FTD, defective FUS likely lacks inhibitory control over deadenylase PAN2, leading to the destabilization and premature


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 56

degradation of mRNAs such as GluA1. C. Defective translocation in SMA. SMN defects inhibit mRNP formation and subsequent β-actin mRNA axonal translocalization, preventing localized translation of β-actin. Such defective trafficking causes neurotransmitting defects due to decreased structural integrity. D. Defective translation in cancer. Upon high translational demand, ribosomal proteins leave to perform translation, abandoning their normal MDM2 sequestration target. Once free, MDM2 marks tumor suppressor p53 for degradation, inhibiting the desired apoptosis response in diseased cells. E. Defective aggregation in ALS. Hexanucleotide repeat expansions within the C9orf72 gene alter cellular function at both the RNA and protein level, to inhibit SG formation by sequestering necessary proteins such as TDP43 and FUS and disturb nucleocytoplasmic transport by interacting with nucleolar and import proteins, respectively.

Figure 4. Mechanisms, benefits, and drawbacks of RNA-based therapies used to treat RNAprocessing defects within neurodegenerative disease and cancers. A. Synthetic siRNA and miRNA are commonly designed for mRNA inhibition and cleavage. Their silencing efficacies


Page 35 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

rely on processing by the endoribonuclease Dicer B. Antisense oligonucleotides do not require cellular processing to interact with mRNA targets to induce cleavage or alternative splicing but require stabilization to protect from ribonucleases.

For Table of Contents Only/Abstract AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was funded by the Welch Foundation Grant No. F-1756 and the Health Effects Institute (HEI) Grant #4949-RFA14-2/15-3. J.C.G.R. was supported by Administrative Department of Science, Technology and Innovation (COLCIENCIAS) from Colombia and


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 56

Fulbright. A.C. was supported by the National Science Foundation (NSF) Graduate Research Fellowship Program. ABBREVIATIONS RNP, ribonucleoprotein; RBP, RNA-binding protein; snRNP, small nuclear ribonucleoprotein; SMA, spinal muscular atrophy; hnRNP, heterogeneous nuclear ribonucleoprotein; snoRNP, small nucleolar ribonucleoprotein; miRNP, microRNA ribonucleoprotein; mRNP, messenger ribonucleoprotein; PABP, polyA-binding protein; P-body, processing body; SG, stress granules; ALS, amyotrophic lateral sclerosis; FTD/FTLD, frontotemporal lobar degeneration; AD, Alzheimer’s disease; CPSF, cleavage and polyadenylation specificity factor; HD, Huntington’s disease; HTT, poly-glutamine-truncated Huntington gene; DKC, dyskeratosis congenita; hTERC, human telomerase RNA gene; TERT, telomerase protein component; FMRP, Fragile X mental retardation protein; SMN, survival motor neuron protein; FUS, fused in sarcoma; TLS, translocated in liposarcoma; TDP-43, TAR DNA-binding protein 43, PABPN1, nuclear poly(A)binding protein; RRM, RNA-recognition motif; RAGE, receptor for advanced glycation end products; mRAGE, full-length membrane bound RAGE; esRAGE, secretory RAGE; APP, amyloid precursor protein; Aβ, amyloid beta/amyloid-β; HRE, hexanucleotide repeat expansion; C9orf72, chromosome 9 open reading frame 72; PABPC, cytoplasmic poly(A)-binding protein; RANT, repeat-associated non-ATG-dependent translation; MDM2, E3 ubiquitin-protein ligase; RIP-seq,

RNA

immunoprecipitation

coupled

to

sequencing;

CHIP-seq,

chromatin

immunoprecipitation coupled to sequencing; RT-PCR, reverse transcription polymerase chain reaction; siRNA, small interfering RNA; MS, mass spectroscopy; RBNS, RNA Bind-N-Seq; RBFOX2, RNA binding protein fox-1 homolog 2; MBNL1, muscleblind like splicing regulator 1; CELF1, CUGBP Elav-like family member 1; TAF15, TATA-box binding protein associated


Page 37 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

factor 15); ncRNA, non-coding RNA; SiMPull, single molecule pulldown; mTOR, mammalian target of rapamycin; FRET, fluorescence resonance energy transfer; RNAi, RNA interference; miRNA, microRNA; EphA2, tyrosine kinase receptor; SSO, splice-switching oligonucleotide; SOD1, superoxide dismutase REFERENCES 1.

Achsel, T., and Bagni, C. (2016) Cooperativity in RNA–protein interactions: the complex

is more than the sum of its partners, Curr. Opin. Neurobiol. 39, 146-151. 2.

Glisovic, T., Bachorik, J. L., Yong, J., and Dreyfuss, G. (2008) RNA-binding proteins

and post-transcriptional gene regulation, FEBS Lett. 582, 1977-1986. 3.

Mourelatos, Z., Dostie, J., Paushkin, S., Sharma, A., Charroux, B., Abel, L., Rappsilber,

J., Mann, M., and Dreyfuss, G. (2002) miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs, Genes Dev. 16, 720-728. 4.

Ule, J. (2008) Ribonucleoprotein complexes in neurologic diseases, Curr. Opin.

Neurobiol. 18, 516-523. 5.

Liu, Q., Fischer, U., Wang, F., and Dreyfuss, G. (1997) The Spinal Muscular Atrophy

Disease Gene Product, SMN, and Its Associated Protein SIP1 Are in a Complex with Spliceosomal snRNP Proteins, Cell 90, 1013-1021. 6.

Geuens, T., Bouhy, D., and Timmerman, V. (2016) The hnRNP family: insights into their

role in health and disease, Hum. Genet. 135, 851-867. 7.

Rossi, S., Cozzolino, M., and Carri, M. T. (2016) Old versus New Mechanisms in the

Pathogenesis of ALS, Brain Pathol. 26, 276-286. 32 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

8.

Page 38 of 56

Hope, N. R., and Murray, G. I. (2011) The expression profile of RNA-binding proteins in

primary

and

metastatic

colorectal

cancer:

relationship

of

heterogeneous

nuclear

ribonucleoproteins with prognosis, Hum. Pathol. 42, 393-402. 9.

Suzuki, H., Shibagaki, Y., Hattori, S., and Matsuoka, M. (2014) Nuclear TDP-43 causes

neuronal toxicity by escaping from the inhibitory regulation by hnRNPs, Hum. Mol. Genet., 1513-1527. 10. Ling, S.-C., Polymenidou, M., and Cleveland, D. W. (2013) Converging mechanisms in ALS and FTD: Disrupted RNA and protein homeostasis, Neuron 79, 416-438. 11. Keller, B. A., Volkening, K., Droppelmann, C. A., Ang, L. C., Rademakers, R., and Strong, M. J. (2012) Co-aggregation of RNA binding proteins in ALS spinal motor neurons: evidence of a common pathogenic mechanism, Acta Neuropathol. 124, 733-747. 12. Nguyen, T. H. D., Galej, W. P., Bai, X.-c., Savva, C. G., Newman, A. J., Scheres, S. H. W., and Nagai, K. (2015) The architecture of the spliceosomal U4/U6.U5 tri-snRNP, Nature 523, 47-52. 13. Lotti, F., Imlach, W. L., Saieva, L., Beck, E. S., Hao, L. T., Li, D. K., Jiao, W., Mentis, G. Z., Beattie, C. E., McCabe, B. D., and Pellizzoni, L. (2012) A SMN-Dependent U12 Splicing Event Essential for Motor Circuit Function, Cell 151, 440-454. 14. Watkins, N. J., and Bohnsack, M. T. (2012) The box C/D and H/ACA snoRNPs: key players in the modification, processing and the dynamic folding of ribosomal RNA, Wiley Interdisciplinary Reviews RNA 3, 397-414.


Page 39 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

15. Phipps, K. R., Charette, J. M., and Baserga, S. J. (2011) The SSU Processome in Ribosome Biogenesis – Progress and Prospects, Wiley interdisciplinary reviews RNA 2, 1-21. 16. Fallini, C., Rouanet, J. P., Donlin-Asp, P. G., Guo, P., Zhang, H., Singer, R. H., Rossoll, W., and Bassell, G. J. (2014) Dynamics of survival of motor neuron (SMN) protein interaction with the mRNA-binding protein IMP1 facilitates its trafficking into motor neuron axons, Dev. Neurobiol. 74, 319-332. 17. Tadesse, H., Deschênes-Furry, J., Boisvenue, S., and Côté, J. (2008) KH-type splicing regulatory protein interacts with survival motor neuron protein and is misregulated in spinal muscular atrophy, Hum. Mol. Genet. 17, 506-524. 18. Erickson, S. L., and Lykke-Andersen, J. (2011) Cytoplasmic mRNP granules at a glance, J. Cell Sci. 124, 293-297. 19. Bekenstein, U., and Soreq, H. (2013) Heterogeneous nuclear ribonucleoprotein A1 in health and neurodegenerative disease: From structural insights to post-transcriptional regulatory roles, Mol. Cell. Neurosci. 56, 436-446. 20. Ramaswami, M., Taylor, J. P., and Parker, R. (2013) Altered Ribostasis: RNA-Protein Granules in Degenerative Disorders, Cell 154, 727-736. 21. Han, Siew P., Tang, Yue H., and Smith, R. (2010) Functional diversity of the hnRNPs: past, present and perspectives, Biochem. J 430, 379. 22. Protter, D. S. W., and Parker, R. (2016) Principles and Properties of Stress Granules, Trends Cell Biol. 26, 668-679.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 56

23. Cáceres, J. F., and Kornblihtt, A. R. (2002) Alternative splicing: multiple control mechanisms and involvement in human disease, Trends Genet. 18, 186-193. 24. Cho, E.-J., Rodriguez, C. R., Takagi, T., and Buratowski, S. (1998) Allosteric interactions between capping enzyme subunits and the RNA polymerase II carboxy-terminal domain, Genes Dev. 12, 3482-3487. 25. Schoenberg, D. R., and Maquat, L. E. (2009) Re-capping the message, Trends Biochem. Sci 34, 435-442. 26. Anthony, K., and Gallo, J.-M. (2010) Aberrant RNA processing events in neurological disorders, Brain Res. 1338, 67-77. 27. Udagawa, T., Fujioka, Y., Tanaka, M., Honda, D., Yokoi, S., Riku, Y., Ibi, D., Nagai, T., Yamada, K., Watanabe, H., Katsuno, M., Inada, T., Ohno, K., Sokabe, M., Okado, H., Ishigaki, S., and Sobue, G. (2015) FUS regulates AMPA receptor function and FTLD/ALS-associated behaviour via GluA1 mRNA stabilization, Nature Commun. 6, 7098. 28. Fica, S. M., Tuttle, N., Novak, T., Li, N.-S., Lu, J., Koodathingal, P., Dai, Q., Staley, J. P., and Piccirilli, J. A. (2013) RNA catalyses nuclear pre-mRNA splicing, Nature 503, 229-234. 29. Wahl, M. C., Will, C. L., and Lührmann, R. (2009) The Spliceosome: Design Principles of a Dynamic RNP Machine, Cell 136, 701-718. 30. Fu, X.-D., and Ares Jr, M. (2014) Context-dependent control of alternative splicing by RNA-binding proteins, Nat. Rev. Genet. 15, 689-701. 31. Dreyfuss, G., Matunis, M. J., Pinol-Roma, S., and Burd, C. G. (1993) hnRNP Proteins and the Biogenesis of mRNA, Annu. Rev. Biochem 62, 289-321. 35 ACS Paragon Plus Environment

Page 41 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

32. Gallouzi, I.-E., and Steitz, J. A. (2001) Delineation of mRNA Export Pathways by the Use of Cell-Permeable Peptides, Science 294, 1895. 33. Buchan, J. R. (2014) mRNP granules: Assembly, function, and connections with disease, RNA Biol. 11, 1019-1030. 34. Kedersha, N., Stoecklin, G., Ayodele, M., Yacono, P., Lykke-Andersen, J., Fritzler, M. J., Scheuner, D., Kaufman, R. J., Golan, D. E., and Anderson, P. (2005) Stress granules and processing bodies are dynamically linked sites of mRNP remodeling, J. Cell Biol. 169, 871-884. 35. Ramaswami, M., Taylor, J. P., and Parker, R. (2013) Altered “Ribostasis”: RNA-protein granule formation or persistence in the development of degenerative disorders, Cell 154, 10.1016/j.cell.2013.1007.1038. 36. Walsh, M. J., Cooper-Knock, J., Dodd, J. E., Stopford, M. J., Mihaylov, S. R., Kirby, J., Shaw, P. J., and Hautbergue, G. M. (2015) Invited Review: Decoding the pathophysiological mechanisms that underlie RNA dysregulation in neurodegenerative disorders: a review of the current state of the art, Neuropathol. Appl. Neurobiol. 41, 109-134. 37. Kishore, S., and Stamm, S. (2011) Role of Alternative Splicing of the 5-HT2C Receptor in the Prader–Willi Syndrome, In 5-HT2C Receptors in the Pathophysiology of CNS Disease (Di Giovanni, G., Esposito, E., and Di Matteo, V., Eds.), pp 413-427, Humana Press, Totowa, NJ. 38. Craig, D. L., Maren, K. F., Emina, H. H., and Thiruvengadam, A. (2007) RAGE and RAGE Ligands in Cancer, Curr. Mol. Med. 7, 777-789. 39. Frohlich, K. M., Sarachan, K. L., Todd, G. C., Basanta-Sanchez, M., Väre, V. Y. P., and Agris, P. F. (2016) Post-Transcriptional Modifications of RNA: Impact on RNA Function and 36 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 56

Human Health, In Modified Nucleic Acids in Biology and Medicine (Jurga, S., Erdmann, V. A., and Barciszewski, J., Eds.), pp 91-130, Springer International Publishing, Cham. 40. Rose, R. E., Pazos, M. A., 2nd, Curcio, M. J., and Fabris, D. (2016) Global Epitranscriptomics Profiling of RNA Post-Transcriptional Modifications as an Effective Tool for Investigating the Epitranscriptomics of Stress Response, Mol. Cell. Proteomics 15, 932-944. 41. Shan, X., Tashiro, H., and Lin, C. L. (2003) The identification and characterization of oxidized RNAs in Alzheimer's disease, J. Neurosci. 23, 4913-4921. 42. Wang, J.-X., Gao, J., Ding, S.-L., Wang, K., Jiao, J.-Q., Wang, Y., Sun, T., Zhou, L.-Y., Long, B., Zhang, X.-J., Li, Q., Liu, J.-P., Feng, C., Liu, J., Gong, Y., Zhou, Z., and Li, P.-F. (2015) Oxidative Modification of miR-184 Enables It to Target Bcl-xL and Bcl-w, Mol. Cell 59, 50-61. 43. Hong, D., Kurzrock, R., Kim, Y., Woessner, R., Younes, A., Nemunaitis, J., Fowler, N., Zhou, T., Schmidt, J., Jo, M., Lee, S. J., Yamashita, M., Hughes, S. G., Fayad, L., Piha-Paul, S., Nadella, M. V., Mohseni, M., Lawson, D., Reimer, C., Blakey, D. C., Xiao, X., Hsu, J., Revenko, A., Monia, B. P., and MacLeod, A. R. (2015) AZD9150, a next-generation antisense oligonucleotide inhibitor of STAT3 with early evidence of clinical activity in lymphoma and lung cancer, Sci. Transl. Med. 7, 314ra185. 44. Cox, G. A., Zang, J. B., Nosyreva, E. D., Spencer, C. M., Volk, L. J., Musunuru, K., Zhong, R., Stone, E. F., Yuva-Paylor, L. A., Huber, K. M., Paylor, R., Darnell, J. C., and Darnell, R. B. (2009) A Mouse Model of the Human Fragile X Syndrome I304N Mutation, PLOS Genetics 5, e1000758.


Page 43 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

45. Banerjee, A., Apponi, L. H., Pavlath, G. K., and Corbett, A. H. (2013) PABPN1: molecular function and muscle disease, FEBS J. 280, 4230-4250. 46. Sun, Y., Grimmler, M., Schwarzer, V., Schoenen, F., Fischer, U., and Wirth, B. (2005) Molecular and functional analysis of intragenic SMN1 mutations in patients with spinal muscular atrophy, Hum. Mutat. 25, 64-71. 47. Tergaonkar, V. (2015) Catalytically inactive telomerase in oncogenesis, Oncotarget 6, 14725-14726. 48. Fronz, K., Güttinger, S., Burkert, K., Kühn, U., Stöhr, N., Schierhorn, A., and Wahle, E. (2011) Arginine methylation of the nuclear poly (a) binding protein weakens the interaction with its nuclear import receptor, transportin, J. Biol. Chem. 286, 32986-32994. 49. Dormann, D., and Haass, C. (2013) Fused in sarcoma (FUS): An oncogene goes awry in neurodegeneration, Mol. Cell. Neurosci. 56, 475-486. 50. Zou, T., Yang, X., Pan, D., Huang, J., Sahin, M., and Zhou, J. (2011) SMN Deficiency Reduces Cellular Ability to Form Stress Granules, Sensitizing Cells to Stress, Cell. Mol. Neurobiol. 31, 541-550. 51. Battle, D. J., Kasim, M., Yong, J., Lotti, F., Lau, C. K., Mouaikel, J., Zhang, Z., Han, K., Wan, L., and Dreyfuss, G. (2006) The SMN Complex: An Assembly Machine for RNPs, Cold Spring Harbor Symp. Quant. Biol. 71, 313-320. 52. Coady, T. H., and Lorson, C. L. (2011) SMN in spinal muscular atrophy and snRNP biogenesis, Wiley Interdiscip. Rev.: RNA 2, 546-564.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 56

53. Honda, D., Ishigaki, S., Iguchi, Y., Fujioka, Y., Udagawa, T., Masuda, A., Ohno, K., Katsuno, M., and Sobue, G. (2014) The ALS/FTLD-related RNA-binding proteins TDP-43 and FUS have common downstream RNA targets in cortical neurons, FEBS Open Bio 4, 1-10. 54. Orozco, D., and Edbauer, D. (2013) FUS-mediated alternative splicing in the nervous system: consequences for ALS and FTLD, J. Mol. Med. 91, 1343-1354. 55. Lagier-Tourenne, C., and Cleveland, D. W. (2009) Rethinking ALS: The FUS about TDP-43, Cell 136, 1001-1004. 56. Workman, E., Kolb, S. J., and Battle, D. J. (2012) Spliceosomal small nuclear ribonucleoprotein biogenesis defects and motor neuron selectivity in spinal muscular atrophy, Brain Res. 1462, 93-99. 57. Gabanella, F., Butchbach, M. E. R., Saieva, L., Carissimi, C., Burghes, A. H. M., and Pellizzoni, L. (2007) Ribonucleoprotein Assembly Defects Correlate with Spinal Muscular Atrophy Severity and Preferentially Affect a Subset of Spliceosomal snRNPs, PLoS One 2, e921. 58. Zhang, Z., Lotti, F., Dittmar, K., Younis, I., Wan, L., Kasim, M., and Dreyfuss, G. (2008) SMN deficiency causes tissue-specific perturbations in the repertoire of snRNAs and widespread defects in splicing, Cell 133, 585-600. 59. Schroeder, R., Barta, A., and Semrad, K. (2004) Strategies for RNA folding and assembly, Nat. Rev. Mol. Cell Biol. 5, 908-919. 60. Hudson, B. I., Carter, A. M., Harja, E., Kalea, A. Z., Arriero, M., Yang, H., Grant, P. J., and Schmidt, A. M. (2008) Identification, classification, and expression of RAGE gene splice variants, FASEB J. 22, 1572-1580. 39 ACS Paragon Plus Environment

Page 45 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

61. Sakurai, S., Yonekura, H., Yamamoto, Y., Watanabe, T., Tanaka, N., Li, H., Rahman, A. K. M. A., Myint, K.-M., Kim, C.-H., and Yamamoto, H. (2003) The AGE-RAGE System and Diabetic Nephropathy, J. Am. Soc. Nephrol. 14, S259-S263. 62. Koyama, H., Yamamoto, H., and Nishizawa, Y. (2007) RAGE and Soluble RAGE: Potential Therapeutic Targets for Cardiovascular Diseases, Mol. Med. 13, 625-635. 63. Donev, R., Newall, A., Thome, J., and Sheer, D. (2007) A Role for SC35 and hnRNPA1 in the Determination of Amyloid Precursor Protein Isoforms, Mol. Psychiatry 12, 681-690. 64. Liu, X.-Y., Li, H.-L., Su, J.-B., Ding, F.-H., Zhao, J.-J., Chai, F., Li, Y.-X., Cui, S.-C., Sun, F.-Y., Wu, Z.-Y., Xu, P., and Chen, X.-H. (2015) Regulation of RAGE splicing by hnRNP A1 and Tra2β-1 and its potential role in AD pathogenesis, J. Neurochem. 133, 187-198. 65. Arnold, E. S., Ling, S.-C., Huelga, S. C., Lagier-Tourenne, C., Polymenidou, M., Ditsworth, D., Kordasiewicz, H. B., McAlonis-Downes, M., Platoshyn, O., Parone, P. A., Da Cruz, S., Clutario, K. M., Swing, D., Tessarollo, L., Marsala, M., Shaw, C. E., Yeo, G. W., and Cleveland, D. W. (2013) ALS-linked TDP-43 mutations produce aberrant RNA splicing and adult-onset motor neuron disease without aggregation or loss of nuclear TDP-43, Proc. Natl. Acad. Sci. U. S. A. 110, E736-E745. 66. Romano, M., Buratti, E., Romano, G., Klima, R., Del Bel Belluz, L., Stuani, C., Baralle, F., and Feiguin, F. (2014) Evolutionarily Conserved Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A/B Proteins Functionally Interact with Human and Drosophila TAR DNA-binding Protein 43 (TDP-43), J. Biol. Chem. 289, 7121-7130.


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 46 of 56

67. Mohagheghi, F., Prudencio, M., Stuani, C., Cook, C., Jansen-West, K., Dickson, D. W., Petrucelli, L., and Buratti, E. (2015) TDP-43 functions within a network of hnRNP proteins to inhibit the production of a truncated human SORT1 receptor, Hum. Mol. Genet. 68. Masuda, A., Takeda, J.-i., Okuno, T., Okamoto, T., Ohkawara, B., Ito, M., Ishigaki, S., Sobue, G., and Ohno, K. (2015) Position-specific binding of FUS to nascent RNA regulates mRNA length, Genes Dev. 29, 1045-1057. 69. Batra, R., Charizanis, K., Manchanda, M., Mohan, A., Li, M., Finn, D. J., Goodwin, M., Zhang, C., Sobczak, K., Thornton, C. A., and Swanson, M. S. (2014) Loss of MBNL Leads to Disruption of Developmentally Regulated Alternative Polyadenylation in RNA-Mediated Disease, Mol. Cell 56, 311-322. 70. Zhang, K., Donnelly, C. J., Haeusler, A. R., Grima, J. C., Machamer, J. B., Steinwald, P., Daley, E. L., Miller, S. J., Cunningham, K. M., Vidensky, S., Gupta, S., Thomas, M. A., Hong, I., Chiu, S.-L., Huganir, R. L., Ostrow, L. W., Matunis, M. J., Wang, J., Sattler, R., Lloyd, T. E., and Rothstein, J. D. (2015) The C9orf72 repeat expansion disrupts nucleocytoplasmic transport, Nature 525, 56-61. 71. Freibaum, B. D., Lu, Y., Lopez-Gonzalez, R., Kim, N. C., Almeida, S., Lee, K.-H., Badders, N., Valentine, M., Miller, B. L., Wong, P. C., Petrucelli, L., Kim, H. J., Gao, F.-B., and Taylor, J. P. (2015) GGGGCC repeat expansion in C9ORF72 compromises nucleocytoplasmic transport, Nature 525, 129-133. 72. Rossi, S., Serrano, A., Gerbino, V., Giorgi, A., Di Francesco, L., Nencini, M., Bozzo, F., Schininà, M. E., Bagni, C., Cestra, G., Carrì, M. T., Achsel, T., and Cozzolino, M. (2015)


Page 47 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Nuclear accumulation of mRNAs underlies G4C2‐repeat‐induced translational repression in a cellular model of C9orf72 ALS

Defective Ribonucleoproteins, Mistakes in RNA Processing, and

Recommend Documents