Viewpoint Cite This: Biochemistry XXXX, XXX, XXX−XXX
pubs.acs.org/biochemistry
High-Throughput Screens for cis-Acting RNA Sequence Elements That Promote Nuclear Retention Kathi Zarnack*,† and Michaela Müller-McNicoll*,‡ †
Buchman Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt, Max-von-Laue-Straße 15, 60438 Frankfurt am Main, Germany ‡ Institute of Cell Biology and Neuroscience, Goethe University Frankfurt, Max-von-Laue-Straße 13, 60438 Frankfurt am Main, Germany
E
Implementing a robust statistical framework, Shukla et al. identified 109 RNA segments that triggered a significant nuclear enrichment. These “differential regions” (DRs) in 29 of the 38 lncRNAs included known RNA localization elements for the lncRNAs MALAT1 and XIST and several novel regions. Nuclear-retained lncRNAs contained more DR copies than cytosolic lncRNAs, and lncRNAs lacking DRs were predominantly cytoplasmic. Similarly, Lubelsky and Ulitsky identified 19 high-confidence regions from 14 genes that conferred at least 30% nuclear enrichment. Within these regions, they identified a cytosine-rich element, RCCTCCC (R = A/G), derived from antisense Alu elements, which was named the SINE-derived nuclear RNA localization element (SIRLOIN). Interestingly, a similar cytosine-rich motif was also overrepresented in many DRs of Shukla et al. By analyzing RNASeq data from the ENCODE project, both studies found that lncRNAs and mRNAs containing the C-rich motif were nuclear-enriched. Testing diverse reporter variations indicated that three SIRLOIN repeats caused stronger nuclear enrichment than a single element,2 while individual C-rich motifs were not able to confer nuclear localization.3 Shuffled SIRLOIN sequences or single-point mutations to purines (A/G) abolished nuclear retention, whereas C → T mutations had little effect.2 To search for trans-acting retention factors, Lubelsky and Ulitsky screened enhanced CLIP (eCLIP) data sets of 112 RNA-binding proteins (RBPs) (ENCODE project). Strikingly, the multifunctional RBP heterogeneous nuclear ribonucleoprotein K (HNRNPK) showed the most binding at SIRLOINs. Consistent with the requirement of three SIRLOIN repeats, HNRNPK harbors three RNA-binding KH domains that cooperatively bind triplets of C/T-rich regions. Moreover, its binding preference closely resembled the RCCTCCC consensus. In line with HNRNPK acting as a global retention factor, eCLIP data demonstrated a strong correlation between HNRNPK binding and nuclear retention. In addition, HNRNPK knockdown decreased the level of nuclear retention for 283 mRNAs with HNRNPK binding sites, and tethering of HNRNPK to a reporter mRNA was sufficient to confer nuclear retention.2 In combination, both studies demonstrate the utility of highthroughput screens for the identification of functional RNA elements and provide substantial evidence that HNRNPK drives the nuclear retention of RNAs by SIRLOIN elements
ukaryotic gene expression is compartmentalized into nuclear and cytoplasmic mRNA processing events, which are connected through the export of mature mRNAs through the nuclear pore. Localization to different cellular compartments influences the availability of protein-coding mRNAs for ribosomes or RNA-degrading enzymes, thereby determining the time, space, and extent of protein production. Recent genome-wide studies identified a wealth of nuclearretained RNAs (reviewed in ref 1). Some retained mRNAs are released into the cytoplasm upon stress or viral infection, suggesting that nuclear retention and release are tightly controlled. Despite being processed by the same machineries, nuclear-retained RNAs have very slow export kinetics, indicating that specific mechanisms inhibit their export. One possibility would be that these RNAs contain specific cis-acting RNA sequence elements that are bound by retention factors to physically anchor them to structural entities or to actively prevent the recruitment of export factors. However, the nature and function of these cis-acting RNA sequence elements are so far poorly understood. Known transcript features that trigger nuclear retention are detained introns, extended poly(A) tails, double-stranded RNA regions, and adenosine-to-inosine editing (reviewed in ref 1). At the RNA sequence level, however, only very few cis-acting elements have been reported, such as the pentamer AGCCC, or short, closely spaced C/T-containing tracts.1 These examples raised the question of whether a universal nuclear localization signal exists within nuclear-retained RNAs. To identify potential nuclear localization signals, two recent studies set up high-throughput screens for cis-acting RNA sequence elements that promote nuclear retention.2,3 Both approaches build on libraries of short oligonucleotides tiling across dozens of candidate RNAs. Shukla et al. designed 11.969 oligonucleotides (110 nucleotides) to cover 38 human long noncoding RNAs (lncRNAs) with nuclear and cytosolic localization.3 Lubelsky and Ulitsky followed a similar scheme with 5.511 oligonucleotides (109 nucleotides) spreading 37 human lncRNAs, 13 3′ untranslated regions of nuclear-enriched mouse mRNAs, and four homologues of MALAT1.2 The oligonucleotides were inserted together with unique molecular identifiers (UMIs) into plasmids carrying efficiently exported reporter genes (Figure 1) and transfected as a pool into human cells. To estimate nuclear retention, the cell lysates were fractionated, and isolated RNA was subjected to targeted RNA sequencing (RNA-Seq) to quantify UMI enrichment in the nucleus. © XXXX American Chemical Society
Received: April 25, 2018
A
DOI: 10.1021/acs.biochem.8b00479 Biochemistry XXXX, XXX, XXX−XXX
Viewpoint
Biochemistry
Figure 1. High-throughput screens for nuclear localization signals. Schematic workflow illustrating major steps common to both approaches (left) and specific differences (right). Abbreviations: pA, poly(A) tail; FDR, false discovery rate; Nuc, nucleus; Cyt, cytoplasm; UTR, untranslated region.
■ ■
and other C-rich motifs. Because SIRLOINs originate from Alu elements, Alu exonization may have provided novel HNRNPKbinding sites to enhance nuclear retention during human transcriptome evolution.4 But how does HNRNPK accomplish this? Notably, the nuclear-retained architectural lncRNA NEAT1, which nucleates membrane-less organelles called paraspeckles, seemed to be excluded from SIRLOIN-mediated retention,2,3 suggesting that independent pathways may exist for distinct RNA classes. However, HNRNPK binds to NEAT1 and is an essential paraspeckle protein; thus, it might be NEAT1 and paraspeckles that sequester HNRNPK along with bound mRNAs or lncRNAs to retain them in the nucleus. In turn, release of bound RNAs in changing cellular conditions might be achieved, e.g. through changes in NEAT1 expression levels or through a displacement of HNRNPK by export adapters, such as SRSF3, which also bind C-rich motifs on mRNAs.5 Further studies are required to unveil this fascinating novel layer of posttranscriptional control of gene expression.
■
ACKNOWLEDGMENTS The authors thank J. König and M. Brüggemann for help with the figure. REFERENCES
(1) Wegener, M., and Müller-McNicoll, M. (2017) Nuclear retention of mRNAs - quality control, gene regulation and human disease. Semin. Cell Dev. Biol., n/a. (2) Lubelsky, Y., and Ulitsky, I. (2018) Sequences enriched in Alu repeats drive nuclear localization of long RNAs in human cells. Nature 555, 107−111. (3) Shukla, C. J., McCorkindale, A. L., Gerhardinger, C., Korthauer, K. D., Cabili, M. N., Shechner, D. M., Irizarry, R. A., Maass, P. G., and Rinn, J. L. (2018) High-throughput identification of RNA nuclear enrichment sequences. EMBO J. 37, e98452. (4) Zarnack, K., König, J., Tajnik, M., Martincorena, I., Eustermann, S., Stevant, I., Reyes, A., Anders, S., Luscombe, N. M., and Ule, J. (2013) Direct competition between hnRNP C and U2AF65 protects the transcriptome from the exonization of Alu elements. Cell 152, 453−466. (5) Müller-McNicoll, M., Botti, V., de Jesus Domingues, A. M., Brandl, H., Schwich, O. D., Steiner, M. C., Curk, T., Poser, I., Zarnack, K., and Neugebauer, K. M. (2016) SR proteins are NXF1 adaptors that link alternative RNA processing to mRNA export. Genes Dev. 30, 553− 566.
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] *E-mail:
[email protected] ORCID
Michaela Müller-McNicoll: 0000-0002-7174-8310 Funding
The authors are grateful for funding from the Deutsche Forschungsgemeinschaft (CRC902 TP13) to K.Z. and M.M.M. Notes
The authors declare no competing financial interest. B
DOI: 10.1021/acs.biochem.8b00479 Biochemistry XXXX, XXX, XXX−XXX
