Orientation of human Microprocessor on primary microRNAs

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Orientation of human Microprocessor on primary microRNAs Huong Minh Nguyen, Trung Duc Nguyen, Thuy Linh Nguyen, and Tuan Anh Nguyen Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00944 • Publication Date (Web): 27 Nov 2018 Downloaded from http://pubs.acs.org on November 29, 2018

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Biochemistry

Orientation of human Microprocessor on primary microRNAs Huong Minh Nguyen1, Trung Duc Nguyen2, Thuy Linh Nguyen2, Tuan Anh Nguyen2,* 1Laboratory of Molecular Microbiology, Institute of Biotechnology, Vietnam Academy of Science and Technology, Hanoi, Vietnam 2Division of Life Science, Hong Kong University of Science & Technology, Hong Kong, China * To whom correspondence should be addressed to Tuan Anh Nguyen. Tel: +852 34692679; Fax: +852 23581552; Email: [email protected]

ABSTRACT Single-stranded microRNAs (miRNAs) regulate gene expression by triggering mRNA degradation and/or inhibiting mRNA translation. miRNAs play important roles in various critical cellular processes and are associated with numerous human diseases, including cancer and neurodegenerative diseases. miRNA sequences are embedded in the primary precursor RNAs (pri-miRNAs) that are initially processed by the Microprocessor complex in the nucleus. Microprocessor can orient itself on pri-miRNAs in two ways: one orientation results in subsequent miRNA production and the other leads to cleavage of the miRNA sequence. Therefore, orienting Microprocessor on pri-miRNAs is a fundamental mechanism to determine the accuracy and efficiency of pri-miRNA processing and, in turn, miRNA production. Multiple mechanisms controlling Microprocessor orientation on pri-miRNAs, involving both cis-acting RNA elements and trans-acting factors, have recently been revealed. In this review, we discuss these exciting mechanisms and consider potential unknown mechanisms that might regulate Microprocessor orientation on pri-miRNAs. Overview of microRNA biogenesis MicroRNAs (miRNAs) are non-coding small RNAs that are approximately 21–22 nucleotides (nt) long. In humans, about 2500 miRNAs have been identified up to date, according to mirbase (mirbase.org). miRNAs function as guide molecules for Ago proteins to find the target mRNAs 13. Often, miRNAs use the region between the 2nd and 8th nucleotide from their 5'-end, namely, the seed sequence, to base pair with the 3'-untranslated region of mRNAs 1-3. The miRNA, Ago, and mRNA form a core of the miRNA-mediated RNA silencing complex that may repress translation and accelerate target mRNA degradation 1-5. Through the silencing of gene expression, miRNAs function in most vital cellular processes and have been associated with various human diseases 6, 7. The majority of miRNAs are canonical miRNAs that are produced in a canonical miRNA biogenesis pathway 8. In this process, primary miRNA transcripts (pri-miRNAs) are synthesized in the nucleus by RNA polymerase II. Although pri-miRNAs are markedly diverse in sequence, they share several common structural features important for their processing (Figure 1A). PrimiRNAs acquire an imperfect stem structure of approximately 3 helical turns of double-stranded RNA (dsRNA), which is flanked by a terminal loop at the top (apical loop) and two single-stranded RNA (ssRNA) segments at the base (basal segments) 9. They also have several common primary sequence features, such as a UG motif at the basal junction, an apical UGU motif at the 5' end of the terminal loop, a CNNC motif at the 3' basal segment, and a GHG motif at the lower stem 10-12. Mature miRNA sequences, the ssRNA regions embedded in pri-miRNAs, are extracted from primiRNAs through two cleavage processes. First, pri-miRNAs are cleaved in the nucleus by Microprocessor to release the intermediate stem-loop RNAs, called pre-miRNAs. The premiRNAs are then exported to the cytoplasm, where they are further cleaved by DICER to generate

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small duplex RNAs 8. The duplex RNAs are taken up by Ago that subsequently keeps only one of the strands as mature miRNAs, while discarding the other 8. It is also suggested that some primiRNAs can be exported to the cytoplasm and there both cleavage reactions take place 13-16.

Figure 1. Pri-miRNA and human Microprocessor. (A) Schematic diagram of a representative primiRNA structure. miRNA–miRNA* duplex is colored in red. The arrows indicate the DROSHA's cleavage sites. The positions of the four motifs, UG, UGU, GHG and CNNC, are illustrated in the figure. The basal UG motif is at basal junction, 14 nt from the DROSHA's cleavage site; the apical UGU motif is at the apical junction, 22 nt from the DROSHA's cleavage site; the GHG motif is at the lower stem, 4 nt from the DROSHA's cleavage site; the basal CNNC motif is at the basal segment, 17 nt from the DROSHA's cleavage site. (B) Human Microprocessor complex is composed of one DROSHA and a DGCR8 dimer. P-rich: Proline-rich domain, R/S-rich: Arginine/Serine rich domain, CED: central domain, RIIIDa and RIIIDb: RNase III domains, dsRBD: dsRNA-binding domain, Rhed: RNA-binding heme domain, CTT: C-terminal tail region. Microprocessor in miRNA biogenesis Microprocessor was discovered and named in 2004 by several groups 17-20. It is a protein complex containing DROSHA and DGCR8 (also called Pasha in Drosophila), both of which are highly conserved through evolution (Figure 1B). Microprocessor functions in a trimeric form composed of one DROSHA and a DGCR8 dimer 12, 21, 22. DROSHA, an RNase III enzyme, is a catalytic subunit that cleaves RNA duplexes, while the DGCR8 dimer is a DROSHA-stabilizing and specific RNA-binding subunit that enhances both the efficiency and accuracy of DROSHA 12, 23-25. Microprocessor functions in multiple RNA metabolic processes, including miRNA biogenesis, rRNA biogenesis, and mRNA degradation 8, 26-30. Dysregulation of the Microprocessor activity is associated with human diseases such as Wilms tumors 31, 32, cancer 33, fragile X-associated tremor/ataxia syndrome 34, and Parkinson's disease 35. DROSHA, a 159 kDa nuclear protein, consists of N-terminal proline-rich and arginine/serine-rich domain, a functionally unknown central domain (CED), and two RNase III domains (RIIIDs), followed by a C-terminal dsRNA-binding domain (dsRBD) (Figure 1B). The N-terminal domain contains the nuclear localization signal (NLS) and various posttranslational modification sites 20.


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Biochemistry

In vivo, this domain is critical for nuclear localization of DROSHA, since the N-terminal truncated DROSHA fragments are not imported into the nucleus and as a result fail to process pri-miRNAs. However, this domain is dispensable for in vitro pri-miRNA processing activity 12, 19. The central domain, corresponding to residues 391 to 941, is essential for the catalytic activity 19. It is suggested that CED might recognize the pri-miRNA junction and the basal UG motif 36. Both RIIIDs and dsRBD are essential for DROSHA's activity. RIIIDs are known to interact with primiRNA's stem and cleave both strands of the stem 12, 19, 36. DGCR8, an 86 kDa nuclear protein, consists of an N-terminal nuclear localization sequence (NLS), an RNA heme-binding domain (Rhed), two dsRBDs, and a C-terminal tail region (CTT) (Figure 1B). CTT alone is sufficient to interact with and stabilize DROSHA 12, 25, 36. The crystal structure of the DROSHA-CTT complex reveals that there are two CTTs that associate with one DROSHA molecule, and deletion of the CTT region (residues 739-750) from DGCR8 completely abolishes its DROSHA-binding capacity 25. Knockout of CTT by CRISPR-Cas9 from human cells (HCT116 cell line) dramatically reduces miRNA expression 37. Several studies show that two dsRBDs along with the C-terminal tail can bind to DROSHA as well as RNA substrates and stimulate the enzyme to cut pri-mir-16-1 as efficiently as full-length DGCR8 12, 25. The replacement of the DGCR8's dsRBDs with two dsRBDs from a different protein (PACT) still retains this stimulatory effect 12. This implies that dsRBDs may stimulate the DROSHA activity by increasing the overall RNA-binding affinity of the complex. However, though dsRBDs enhance DROSHA cleavage efficiency on pri-mir-30a substrate, the dsRBDs-DROSHA complex fails to cleave primir-30a at the productive site 12. This indicates that, in some cases, dsRBDs alone is not enough to ensure accurate pri-miRNA processing. Recently, extensive biochemical studies using purified DGCR8 proteins revealed that Rhed is a multi-functional domain that contains dimerization, hemin-binding, and RNA-binding capacities 12, 23, 24. Dimerization is a conserved feature among many DGCR8 homologs and contributes to its substrate binding specificity as well as processing efficiency in vitro and in vivo 12, 23, 24, 38-41. The recent studies also showed that DGCR8 also interacts with the loop and might possess an ability to recognize the apical UGU motif 12, 24. Though it is not clear how DGCR8 interacts with the UGU motif, Rhed may possess this feature, since DGCR8 fragments lacking this domain no longer recognize the UGU motif 12. In addition, the highest UGU-binding affinity is only achieved when the DGCR8 dimer associates with hemin 37. However, it was also reported that DGCR8 nonspecifically binds to ssRNAs and dsRNAs with a strong affinity 42, suggesting that the substrate specificity of DGCR8 may be achieved only in presence of DROSHA. Cis-acting elements regulate Microprocessor orientation on pri-miRNAs The clear basal junction Pri-miRNAs have a somewhat symmetric secondary structure. Their stem is a dsRNA of ~35 base pairs (bp). One end of the stem is flanked by two ssRNA segments to make a basal junction, while the other end is connected by a ssRNA apical loop, to create an apical junction. The expected cleavage site of Microprocessor is 11 bp from the basal junction (Figure 1A). It was first proposed that DGCR8 acts as a ruler of Microprocessor by interacting and measuring the distance between the basal junction and cleavage site, thereby locating DROSHA at the basal junction to execute the cleavage 9. Counting 11 bp from the basal junction is called the basal junction rule. The junction rule is further supported by subsequent studies 10, 12. However, it turned out that DROSHA, instead of DGCR8, is the junction ruler (Figure 2A). It indeed has an ability to recognize the junction and position its catalytic centers at 11 bp from the said junction to cleave pri-miRNAs 12, 36.


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Figure 2. The clear basal junction rule. (A) DROSHA recognizes the basal junction and measures 11 bp from this junction to process pri-miRNAs. (B) DROSHA recognizes the apical junction of pri-miRNAs with clear junction structure. The green and purple arrowheads represent the productive and unproductive cleavage sites of DROSHA, respectively. Mature miRNAs are indicated in red region. Since pri-miRNAs have two junctions at two ends of the stem, DROSHA might not be able to distinguish one from the other. In the absence of DGCR8, DROSHA can bind and cleave at both junctions of an artificial pri-miRNA 12, or of pri-mir-142 43, or at an unexpected junction of pri-mir30a 12. It is likely that a clear junction would be recognized and cleaved better by DROSHA (Figure 2A). “A clear junction” is a junction with two ssRNA segments of ~5 nt that do not base pair with each other. DROSHA can efficiently and accurately cleave pri-mir-16-1 at the basal junction. When the clear junction of pri-mir-16-1 is disrupted by introducing a few base pairs between two basal RNA segments, DROSHA significantly loses its cleavage efficiency 12. In consistent with this, DROSHA does not cleave at the basal junction of pri-mir-30a efficiently, since this pri-miRNA does not have a clear basal junction. Instead, DROSHA cleaves pri-mir-30a mainly at the apical junction that is clearer (Figure 2B). The mutations that open a few base pairs between two basal RNA segments of pri-mir-30a allow DROSHA to better interact and cleave at the basal junction. Taken together, the clear junction rule allows DROSHA to find an accurate cleavage position for many pri-miRNA substrates; however, it also leads DROSHA to mis-localize at the apical junction of some other pri-miRNAs (Figure 2B). Therefore, additional orienting mechanisms are needed for various pri-miRNAs (discussed below). The UG motif In order to reinforce the interaction of DROSHA and the basal junction, many pri-miRNAs possess the UG motif that has a binding affinity to DROSHA. The location of the UG motif is at the 5' basal segment and 14 nt from the cleavage sites. In RNA substrates that have two similar junctions, the introduction of the UG motif at either junction results in a biased interaction and cleavage by DROSHA at the UG-containing junction (artificial pri-miRNA, 12) (Figure 3A–B). In pri-mir-16-1, replacement of UG at its basal junction by the CC motif reduces the DROSHA enzyme activity, whereas the relocation of UG from the basal junction to the apical junction leads DROSHA to cleave more at the apical junction 12. A similar effect of the UG motif is also observed with a full complex of Microprocessor. These biochemical observations indicate that the UG motif plays a role in orienting Microprocessor on pri-miRNA through DROSHA (Figure 3A–B). In humans, the UG motif is present in 24.3% of human pri-miRNAs that are conserved with mice, so-called representative pri-miRNAs. Unlike in humans, pri-miRNAs of C. elegans often do not possess the UG motif. However, the introduction of the UG motif to AG motif-containing pri-miRNAs (cel-miR44) of C. elegans could increase their miRNA expression in human cells 10.


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Biochemistry

Figure 3. The UG rule. (A) The UG motif locates at 5' segment of the basal junction and 14 nt from the cleavage site. This basal UG motif recruits DROSHA to the basal junction to process primiRNA productively. (B) Without the UG motif, DROSHA can bind to pri-miRNA either ways and cleave it at the productive and non-productive sites. The UGU motif

Figure 4. The UGU rule. (A) The UGU motif is located in the terminal loop. The DGCR8 dimer recognizes and interacts with this apical UGU motif, thereby preventing DROSHA from binding to this junction. This indirectly forces DROSHA to cleave at the basal junction. (B) Without the UGU motif, pri-miRNAs can be cleaved at both apical and basal sites by Microprocessor. DROSHA alone shows a preference for the apical junction and cleaves at this junction of pri-mir30a. However, in a complex with DGCR8, DROSHA changes its cleavage sites to the basal junction 12. DGCR8 does this by interacting with the apical loop and the apical UGU motif to block DROSHA from binding to the apical junction, thereby correctly locating DROSHA at the basal junction 12 (Figure 4A–B). Replacement of the UGU motif of pri-mir-30a by the AAU motif reduces the accuracy of the pri-mir-30a processing. In addition, the introduction of the UGU motif to the apical junction of the artificial pri-miRNAs stimulates the DGCR8-apical loop interaction, which in turn enhances the Microprocessor function at the basal site 12. This mechanism indirectly orients DROSHA at the basal junction by allowing DGCR8 to occupy the apical junction. Notably, the UGU-recognizing ability of DGCR8 is acquired only when DGCR8 exists in the dimer form. Consistently, the expression of DGCR8 alleles producing the DGCR8 dimer could stimulate the UGU-containing miRNA expression more efficiently than that making the DGCR8 monomer 37. It is implicated that the Rhed domain of DGCR8 might contain the UGU-recognition amino acid


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residues since the DGCR8 fragments lacking this domain are unable to recognize the UGU motif 12. The UGU motif appears in 29.9% of the representative pri-miRNAs 10. The fact that the Microprocessor subunits recognize both UG and UGU motifs suggests that the appearance of these motifs in pri-miRNAs co-evolved with DROSHA and DGCR8 of Microprocessor. The introduction of the UG or UGU motif to pri-miRNAs of C. elegans positively influences their processing in human cells 10. Interestingly, the location of the UG and UGU in the artificial primiRNAs could change the orientation of Microprocessor on pri-miRNAs. The basal UG and apical UGU direct Microprocessor to cut at basal sites, while apical UG and basal UGU completely flip Microprocessor to cleave at the apical sites 12. Transacting factors regulate Microprocessor orientation on pri-miRNAs Hemin

Figure 5. The hemin rule. (A) Hemin promotes the pri-miRNA processing of Microprocessor efficiently and accurately by strengthening the interaction of the DGCR8 dimer and the apical UGU motif. (B) In the absence of hemin, Microprocessor can cleave pri-miRNAs at both orientations. Hemin is a ferric form (Fe3+) of heme that consists of a ferrous ion (Fe2+) at the center of a porphyrin ring and is a critical cofactor for various proteins and enzymes. Hemin enhances the pri-miRNA processing in vitro and in vivo for several tested pri-miRNAs, though its molecular mechanism remains unknown. Recent findings suggest that hemin plays a role in orientation of Microprocessor on pri-miRNAs 37, 44 (Figure 5A–B). One study purified the Microprocessor complex with or without hemin by co-expressing DROSHA and DGCR8 in the insect cell system and tested their activity with several pri-miRNAs 44. In another study, DGCR8-P351A with an alanine mutation at the 351st proline position was investigated 37. This mutation has a weak hemin binding affinity 39. This form could be purified as a dimer without hemin (Apo-DGCR8) from E. coli and reconstituted with synthetic hemin in vitro. Subsequently, the Microprocessor complex was constructed by mixing the purified DROSHA and DGCR8 proteins with or without hemin. Both studies share a similar observation that the trimeric Microprocessor complex changes its orientation on pri-miRNAs upon interaction with hemin. Hemin likely strengthens the interaction between DGCR8 dimer and the apical loop (Figure 5A–B). In addition, hemin enhances the interaction between DGCR8 and the apical UGU motif of pri-mir-30a in vitro, since pri-mir-30a lacking the UGU motif could not be accurately and efficiently processed by Microprocessor even


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Biochemistry

in presence of hemin 37. This also holds true for artificial pri-miRNAs, suggesting that this might be a universal mechanism for the processing of many other pri-miRNAs 37. Consistent with the in vitro findings, hemin differentiates miRNA expression in vivo. It stimulates the expression of UGU-containing miRNAs more efficiently than nonUGU-containing miRNAs in human cells 37. Although it is not known how hemin affects the interaction between DGCR8 and UGU, it may likely change the conformation of the DGCR8 dimer so that DGCR8 acquires a proper structure for RNA interaction. Revealing the architecture of the DGCR8-UGU/hemin complex may shed light on their interaction mechanism. SRSF3 and the CNNC motif

Figure 6. The SRSF3/CNNC rule. (A) SRSF3 interacts with the CNNC motif located at the 3′segment of the basal junction ~16–18 nt from the DROSHA cleavage sites. This interaction contributes to recruitment of DROSHA to the basal junction of pri-miRNAs, thereby correcting the orientation of Microprocessor on pri-miRNAs. Consequently, SRSF3 promotes DROSHA to cleave at the productive sites. (B) In the absence of the CNNC motif, SRSF3 fails to recruit DROSHA to the basal junction; therefore, Microprocessor can process pri-miRNAs in both directions. SRSF3 (SRP20) is a splicing factor that plays essential roles in RNA metabolism 45-57. SRSF3 interacts with the CNNC motif located at the 3' basal segment to stimulate pri-miRNA processing 10, 43, 58. A recent study revealed that SRSF3 could change the orientation of DROSHA on primiRNAs 43 (Figure 6A–B). DROSHA alone binds and cleaves pri-mir-30a at the apical junction; however, the addition of SRSF3 causes DROSHA to cleave pri-mir-30a at the basal junction. This indicates that SRSF3 binds to the CNNC motif at the basal segment and recruits DROSHA from apical junction to the basal junction (Figure 6A–B). Consequently, SRSF3 stimulates the correct orientation of Microprocessor on pri-mir-30a. This effect of SRSF3 is dependent on the presence of the CNNC motif, since SRSF3 fails to attract DROSHA to the basal junction without the CNNC motif. In addition, SRSF3 can shift DROSHA around the expected cleavage sites according to the location of the CNNC motif. The optimal position of CNNC for stimulating the accurate DROSHA' cleavage is 16–18 nt from the cleavage site. This mechanism might partially be the reason why the CNNC motif is highly enriched (in ∼60% of the representative pri-miRNAs) in the basal segment of pri-miRNAs within a narrow window of 16–18 nt from the cleavage site. Prospective Microprocessor orientation-regulating mechanisms The processing of pri-miRNA heavily relies on the direction of Microprocessor on pri-miRNAs. Therefore, orientation mechanism is critical to controlling the miRNA expression level. Multiple


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mechanisms have been discussed in this review. Besides that, unknown mechanisms involving cis-acting and trans-acting factors could be proposed, firstly because, pri-miRNAs have different structures with unknown motifs. Secondly, Microprocessor associates with various cofactors that may regulate the complex under different cellular circumstances. The GHG motif The GHG motif is a string of three nucleotides at positions 7 to 9 from the basal junction of primiRNAs 11. The randomized combinatorial synthesis was used to introduce mutations across the stem of three pri-miRNAs, pri-mir-125, pri-mir-16 and pri-mir-30, and of all positions studied, only base pairing at location 8 shows an adverse effect on DROSHA cleavage efficiency in all three pri-miRNA templates. Further analysis from the authors revealed the full GHG motif includes an enriched paired G at position 7, any other base but G at position 8, and another enriched paired G at position 9. The GHG motif is also called the “mismatched GHG,” noting the fact that H at position 8 is frequently found as a mismatch in pri-miRNA templates where DROSHA shows efficient cleavage. The mismatched GHG motif is conserved in both humans and Drosophila and observed in some other vertebrates. In vitro competitive cleavage assay using pri-mir-125 showed that flipping of the G-C base pairs at either one or both positions of 7 and 9 results in 33%, 26% and 74% loss of DROSHA cleavage efficiency, respectively 11. Introduction of either U-A or U-G pairing at position 8 also decreases DROSHA activity to 66% and 45%, respectively. The GHG motif shows redundancy, with the first introduced GHG motif showing the most substantial effect (up to 12 fold) compared with others in a multi-motif template. In vivo, the addition of a GHG motif to the C. elegans pri-mir-44, which is poorly processed in mammalian cells, improves mature miRNA accumulation by more than two folds. So how does the presence of the mismatched GHG at the basal stem of pri-miRNA substrates help Microprocessor with its processivity and accuracy? One hypothesis is that this motif is recognized by and interacts with DROSHA, thus assisting Microprocessor to correctly orient and position itself on pri-miRNA substrates to carry out productive cleavages (Figure 7B). So far, it remains unclear how Microprocessor recognizes and interacts with the GHG motif. Pri-miRNA modifications Aside from primary sequence and structure features, several studies have reported modifications on pri-miRNAs may play essential roles in miRNA biogenesis (Figure 7B). One of the posttranscriptional modifications found in pri-miRNAs is N6-methyl-adenosine (m6A), a methylated adenosine residue appeared as a GGAC motif in pri-miRNAs. A bioinformatics search revealed that this motif is enriched in human pri-miRNAs, and it serves as the recognition sequence for the RNA methyltransferase enzyme METTL3 59. Methylation of pri-miRNAs at the GGAC motif affects pri-miRNA processing, as shown in the gainof-function experiment using HEK293T cells overexpressing DGCR8 and DROSHA, where methylated templates are processed more efficiently than unmethylated ones. This effect is diminished by METTL3 depletion, with approximately 70% of miRNAs being down regulated by at least 30% in the absence of METTL3, proving the role of METTL3 as the main RNA methylase involved in this process. The direct involvement of METTL3 in pri-miRNA biosynthesis is further validated by the immunoprecipitation experiments showing that METTL3 and DGCR8 coprecipitated in the presence of RNA substrates. This binding is RNA-mediated since METTL3 fails to co-precipitate with DGCR8 when RNase is added to the reaction to degrade RNA moieties. It is still unclear how m6A acts as a marker for enhancing pri-miRNAs processing. Since m6A sites are rather distant from the stem-loop region of pri-miRNAs, it is unlikely that Microprocessor directly recognizes m6A. A more plausible mechanism is that m6A is recognized by an m6A


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reader that binds it and mediates the interaction between Microprocessor and pri-miRNAs, thereby affecting the orientation of Microprocessor. One such likely reader is hnRNP A2/B1, whose depletion significantly reduces the expression of many miRNAs 59. hnRNP A2/B1 regulation of pri-miRNA processing is METTL3-dependent, since deletion of METTL3 affects the expression of about 95% of the hnRNP A2/B1-regulated miRNAs.

Figure 7. Prospective mechanisms of cis-acting elements and trans-acting factors regulating pri-miRNA processing. (A) The current understanding of how Microprocessor and its cofactors (hemin and SRSF3) interact with the different motifs of pri-miRNAs, UG, UGU, and CNNC. (B) The unidentified RNA elements or RNA modifications of pri-miRNAs, such as m6A or A-I modifications might influence the orientation of Microprocessor via changing the interaction between pri-miRNAs and either subunit of Microprocessor. (C) The known and unknown protein factors that interact with pri-miRNAs, or Microprocessor, or both pri-miRNAs and Microprocessor might affect Microprocessor orientation on pri-miRNAs. (D) DROSHA and DGCR8 proteins could be modified. The modified proteins might exhibit different RNA-binding properties, thereby regulating the Microprocessor orientation on pri-miRNAs. In contrast, deletion of hnRNP A2/B1 only reduces about 50% of the METTL3-affected miRNAs, suggesting that hnRNP A2/B1 only regulates about half of METTL3-methylated pri-miRNAs. This may indicate that as yet unknown factors might read the methylation sites of pri-miRNAs. It is suggested that hnRNP A2/B1 recruits DGCR8 to pri-miRNAs, since they display a direct proteinprotein interaction 59. Currently, many studies have pointed out that DGCR8 does not recognize the basal junction of pri-miRNAs 12, 36, and since m6A sites mostly localize at the basal regions of pri-miRNAs, it is more likely that the m6A reader recruits DROSHA instead. Another well-established post-transcriptional regulation of miRNA biogenesis is the deamination of adenosines to inosines by adenosine deaminases (ADARs). This reaction may convert a Watson-Crick pair of A-U into a wobble pair of I-U, thus changing primary RNA sequences and


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secondary dsRNA conformation. Both recombinant ADAR1 and ADAR2 efficiently edit four out of eight tested pri-miRNAs in vitro 60. Editing sites are specific for each enzyme. In the case of primir-142, Microprocessor could produce pre-miRNA from wild-type pri-mir-142 (unedited, canonical Watson-Crick pair A-U), while it fails to do so on pre-edited pri-mir-142 (four G-U or UG pairs instead of A-U) 60. These observations suggest that RNA editing by ADARs might inhibit miRNA maturation at the pri-miRNA processing step. Since ADAR editing changes the stem structure of pri-miRNAs and leads to inhibition of Microprocessor cleavage activity, it is conceivable to hypothesize that ADAR editing interferes with either Microprocessor positioning or orientating on pri-miRNA templates. Trans-acting factors Many RNA-binding proteins (RBPs) have been identified as the miRNA expression regulators 8, 61-65. For examples, the stem cell factor Lin28 interacts with let-7 miRNA family 66-68, and hnRNP A1 functions as DROSHA's auxiliary factor through binding to pri-mir-18a 69, 70. The well-known Lin28/ TUT4(7) plays important roles in regulating the expression of the let-7 miRNA family in stem cells and cancer pathogenesis 71. The human Y box-binding protein YBX1 is known to modulate miRNA processing in glioblastoma multiforme 72. A subset of miRNAs containing an apical loop enriched with a short G-rich stretch, including the let-7 miRNA family, which is important tumor suppressor miRNAs, is dependent on KHSRP for their biogenesis. SUMOylation of KHSRP at K87 blocks its binding to pri-miRNAs and Microprocessor, leading to the impaired biogenesis of the tumor suppressive let-7 family miRNAs 71. Most recently, using a proteomicsbased high throughput pull-down approach, Treiber and colleagues built the interactome map of multiple pri-miRNAs, and characterized many novel RBPs regulating miRNA biogenesis 65. The notable newly identified RBPs that modulate miRNA expression include ZC3H10 and CELF1/2 (stem-binding proteins), TRIM71 (apical loop-binding protein), and hnRNP A0, hnRNP A2/B1, and hnRNP A3. It is interesting to see if they regulate the orientation or position of Microprocessor on pri-miRNA templates (Figure 7C). Below we discuss two factors, DDX17 and hnRNP A1, as examples in more detail. DDX17 is an RNA helicase enzyme that unwinds dsRNAs, thus playing essential roles in multiple cellular processes. Its involvement in various steps of human RNA metabolism has made it a prominent target for numerous cellular regulation mechanisms and pathogenesis. DDX17 is a member of the DEAD box RNA helicase family that contains the conserved DExD motif. DDX17 acts as one of the accessory proteins of the Microprocessor complex. A recent study revealed that sequestering of DDX17 by transcriptional coactivator YAP results in inhibition of the Microprocessor activity and eventually leads to tumorigenesis 73. It was first observed that miRNA level is elevated at high cell density in a YAP-dependent manner. When the authors looked at the expression level of DROSHA and DGCR8, their expressions are not altered, suggesting that cell density changes Microprocessor's activity. Co-immunoprecipitation revealed that YAP directly interacts with DDX17, thus preventing DDX17 from interacting with the Microprocessor components, eventually leading to a global decrease in miRNA level and accumulation of primiRNAs. This indicates that pri-miRNA processing is defective in these cancer cells and likely the causative factor for tumorigenesis. The mechanism by which DDX17 interacts with Microprocessor remains mostly unknown. Truncation experiments showed that deletion of the pri-miRNA's stem-loop and 5' basal segment does not significantly affect this interaction, but deletion of the 3' basal segment almost completely abolishes DDX17 binding to Microprocessor. A search for an overrepresented sequence motif in the 3' basal segment revealed a VCAUCH motif. In mutant pri-miRNAs where this motif is disrupted, Microprocessor interaction with DDX17 is significantly impaired, suggesting that


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Biochemistry

DDX17 selectively binds to this motif to interact with Microprocessor and enhance its activity. Based on the VCAUCH position in the 3' basal segment, the presence of DDX17 at the VCAUCH motif in the 3' basal segment might correctly position and orient DROSHA on pri-miRNAs, thus enhancing Microprocessor processing efficiency. It recently comes to light that SWI2/SNF2 ATPase regulates the Microprocessor activity in plants by changing the secondary structure of pri-miRNAs 74. Thus, it is possible that DDX17 might also remodel pri-miRNA's secondary structures using its helicase activity, thereby altering the Microprocessor activity. Further biochemical experiments are needed to examine the molecular mechanism of DDX17 on primiRNA processing. Another trans-acting factor that directly binds to pri-miRNAs is the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), a member of the diverse family of RBPs. In vivo, hnRNP A1 binds to the apical loop of human pri-mir-18a and induces partial relaxation of its stem, thus aiding efficient DROSHA cleavage of pri-mir-18a 69. A recent study illustrates that hnRNP A1 utilizes its two RNA-recognition-motif domains to interact with two UAG motifs in the apical loop and proximal stem region of pri-mir-18a 75. This binding event induces the unwinding of the target stem-loop region. The authors proposed three possible mechanisms in which hnRNP A1 can aid pri-mir-18a processing: 1) influence stem destabilization thus enhance DROSHA cleavage efficiency, 2) promote the correct orientation of Microprocessor on pri-mir-18a by generating a thermodynamic or conformational asymmetry in the stem, 3) increase the accessibility of Microprocessor to the pri-mir-18a hairpin in the context of the complete cluster. In contrast, hnRNP A1 negatively regulates the expression of let-7a by binding to their apical loop 70. The binding site of hnRNP A1 at the conserved loop of pri-let-7a is found to overlap with that of the splicing regulatory protein KSRP, which promotes let-7a biogenesis 76. It is interesting to explore how hnRNP A1 confers the opposite effects on the Microprocessor activity. One likely explanation for this phenomenon might be that differences in hnRNP A1 binding sites might favor different orientation and position of Microprocessor on pri-miRNAs, eventually resulting in different cleavage efficiency. Protein modifications Microprocessor is subjected to various post-translational modifications that regulate and fine tune their various cellular functions (Figure 7D). Post-translational modifications affect Microprocessor localization, stability, and enzyme activity. For example, phosphorylation is needed for correct localization of DROSHA to the nucleus 77, and for the stabilization of DGCR8 78. Acetylation of the N-terminus of DROSHA helps inhibit its degradation by ubiquitination 79. DGCR8 affinity for primiRNAs is increased by its deacetylation by HDAC1 80, leading to a general upregulation of miRNA level. SUMOylation at the K707 site of DGCR8 by SUMO1 increases DGCR8 stability and affinity to pri-miRNAs but neither alters its interaction with DROSHA nor improves miRNA biogenesis 81. It is necessary to understand how two modifications increase DGCR8 affinity to primiRNA, but while one boosts miRNA biogenesis, one does not. Conclusion and Outlook In this review, we summarize the known mechanisms regulating the orientation of Microprocessor on its substrates, pri-miRNAs. This seems to be an essential mean that controls subsequent miRNA maturation. We also discuss several identified factors and RNA modifications that are associated with pri-miRNA processing in multiple ways and suggest their roles in Microprocessor orientation on pri-miRNAs. We also discuss as yet mechanisms that could potentially play a role in regulating Microprocessor orientation. It is essential to figure out various molecular mechanisms to understand how miRNA expression is differentiated in multiple cellular contexts, such as in human diseases. The obtained knowledge is also necessary for designing artificial primiRNAs for application in knockdown technology.


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Biochemistry

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Orientation of human Microprocessor on primary microRNAs

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