High-throughput identification of RNA nuclear enrichment sequences

Abstract

Summary 27One of the biggest surprises since the sequencing of the human genome has been the discovery 28 of thousands of long noncoding RNAs (lncRNAs) [1][2][3][4][5][6] . Although lncRNAs and mRNAs are similar 29 in many ways, they differ with lncRNAs being more nuclear-enriched and in several cases 30 exclusively nuclear 7,8 . Yet, the RNA-based sequences that determine nuclear localization remain 31 poorly understood [9][10][11] . Towards the goal of systematically dissecting the lncRNA sequences that 32 impart nuclear localization, we developed a massively parallel reporter assay (MPRA). Unlike 33 previous MPRAs 12-15 that determine motifs important for transcriptional regulation, we have 34 modified this approach to identify sequences sufficient for RNA nuclear enrichment for 38 human 35 lncRNAs. Using this approach, we identified 109 unique, conserved nuclear enrichment regions, 36originating from 29 distinct lncRNAs. We also discovered two shorter motifs within our nuclear 37 enrichment regions. We further validated the sufficiency of several regions to impart nuclear 44RNA subcellular localization provides a fundamental mechanism through which cells modulate 45 and utilize the functions encoded in their transcriptomes 16 . This spatial layer of gene regulation is 46 known to be critical in a variety of contexts, including asymmetric cell divisions 17 , embryonic 47 development [18][19][20] , and signal transduction 21 . Previous work has identified a small number of cis-48 acting mRNA localization elements, using genetic approaches or hybrid reporter constructs to 49 decipher sequences required for localization to different parts of the cell 16,18 . These elements are 50. CC-BY-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/189654 doi: bioRxiv preprint first posted online Sep. 15, 2017; often located in 3´ untranslated regions (UTRs), and range from five to several hundred 51 nucleotides in length [9][10][11]18 . Yet, the sequences and structures responsible for RNA localization 52 remain inchoate. In contrast to mRNAs that are mostly localized outside the nucleus, lncRNAs 53 are enriched or retained in the nucleus. Increasing evidence suggests that many lncRNAs may 54 reside in the nucleus for the purpose of regulating nuclear processes, including formation of 55 paraspeckles, topological organization of the nucleus, and regulation of gene expression 1,3,4,22 . 56However, while it is now evident that lncRNAs have important functions in the nucleus 22 , very little 57 is known about specific sequence elements driving their nuclear enrichment 9-11 . 59To elucidate which sequences drive lncRNA nuclear enrichment, we developed a high-throughput 60 approach for identifying nuclear enrichment elements. Our approach, derived from a massively 61 parallel reporter assay (MPRA) [12][13][14][15] 23 , is based on a previous assay demonstrating that the native 62 cytosolic loc...

Nuclear sorting of RNA

“…Retention of MALAT1 in speckles depends on two distinct sequence elements, which are also involved in binding to the nuclear speckle enriched protein RNPS1 (Figure ; Miyagawa et al, ). Consistently, these regions were identified in a recent high‐throughput screen for RNA sequence elements that promote nuclear retention (Shukla et al, ).…”

“…The Massively Parallel RNA assay (MPRNA) by Shukla et al () took advantage of the sequences of 37 lncRNAs, with known localizations in both nuclear and cytoplasmic compartments, to generate approximately 12,000 overlapping barcoded oligonucleotides. These sequences were then fused to reporter constructs, known for their native cytoplasmic localizations, and transfected into HeLa cells before sequencing either nuclear or total cellular RNA (Shukla et al, ). Approximately 110 unique RNA tags were identified, that showed a significant relative enrichment in the nuclear fraction.…”

“…Approximately 110 unique RNA tags were identified, that showed a significant relative enrichment in the nuclear fraction. Subsequent motif analysis of the putative retention signals showed the enrichment of cytosine rich sequences (Shukla et al, ). A parallel study from Lubelsky and Ulitsky () utilized a similar methodology with a tiling‐based screen of lncRNAs, 3′ UTRs of nuclear enriched mRNAs and homologs of MALAT1 .…”

No way out: when RNA elements promote nuclear retention

“…Two new studies now use elegant tiling‐based approaches to screen for cis ‐acting RNA elements involved in nuclear retention. The first study (Shukla et al , ) developed Massively Parallel RNA Assay (MPRNA) that is based on sequences from 38 human lncRNAs, which were chosen to cover a spectrum of nuclear and cytosolic localization patterns. Briefly, 11.969 unique, barcoded oligos of 153 nt length were designed to tile the lncRNA sequences in a partially overlapping manner (Fig ).…”

“…The study by Lubelsky and Ulitsky () found that both the presence of SIRLOIN motifs and the presence of hnRNPK binding sites correlate with nuclear enrichment of lncRNAs and mRNAs. Shukla et al () performed a comparative analysis of ENCODE RNA‐seq data produced from nuclear and cytoplasmic fractions of 11 cell lines and similarly found a correlation of DRs with nuclear retention, with the effect being more pronounced for lncRNAs than mRNAs. Interestingly, the RNAs that are strictly nuclear tend to have many repeats of DRs; for example, 18 DRs are present in XIST and 10 in MALAT1 lncRNAs.…”