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HIGH-THROUGHPUT IDENTIFICATION OF RNA LOCALIZATION ELEMENTS REVEALS A REGULATORY ROLE FOR A/G RICH SEQUENCES

ABSTRACT: Hundreds of RNAs are enriched in the projections of neuronal cells. For the vast majority of them, though, the sequences within them that regulate their localization are unknown. To identify RNA elements capable of directing transcripts to neurites, we designed and deployed a massively parallel reporter assay that tested the localization regulatory ability of thousands of sequence fragments drawn from endogenous 3′ UTRs. We identified peaks of regulatory activity within several 3′ UTRs and found that sequences derived from these peaks were both necessary and sufficient for RNA localization to neurites. These active localization elements were rich in adenosine and guanosine residues. They were also required to be dozens of nucleotides long as subsequences showed significantly reduced activity. Using RNA affinity and mass spectrometry, we found that the RNA-binding protein Unk was associated with the active localization elements. Depletion of Unk in cells reduced the neurite-enrichment of the active localization, indicating a functional requirement for Unk in their trafficking. These results provide a framework for the unbiased, high-throughput identification of RNA elements and mechanisms that govern transcript localization.

ORGANISM(S): Mus musculus

PROVIDER: GSE183192 | GEO | 2021/09/03

REPOSITORIES: GEO

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Project description:Much of posttranscriptional mRNA regulation occurs through cis-acting sequences in mRNA 3´ untranslated regions (UTRs), which interact with specific proteins and ribonucleoprotein complexes that modulate translation, mRNA stability and subcellular localization. Studies in Caenorhabditis elegans have revealed indispensable roles for 3´UTR-mediated gene regulation, yet most C. elegans genes have lacked annotated 3´UTRs. Here we describe a high-throughput method to reliably identify 3´ ends of polyadenylated RNAs. This method, called poly(A)-position profiling by sequencing (3P-Seq), was used to determine the UTRs of C. elegans. Compared to standard methods also recently applied to C. elegans UTRs, 3P-Seq identified 8775 additional UTRs while excluding thousands of shorter UTR isoforms that do not appear to be authentic. Analysis of this expanded and corrected dataset indicated that the high A/U content of C. elegans 3´UTRs facilitated genome compaction, since the elements specifying cleavage and polyadenylation, which are A/U-rich, can more readily emerge in A/U rich regions. Indeed, 30% of the protein-coding genes have mRNAs with alternative, partially overlapping end regions that generate another 10,000 cleavage and polyadenylation sites that had gone largely unnoticed and represent potential evolutionary intermediates of progressive UTR shortening. Moreover, a third of the convergently transcribed genes utilize palindromic arrangements of bidirectional elements to specify UTRs with convergent overlap, which also contributes to genome compaction by eliminating regions between genes. Although nematode 3´UTRs have median length only one-sixth that of mammalian 3´UTRs, they have twice the density of conserved microRNA sites, in part because additional types of seed-complementary sites are preferentially conserved. These findings reveal the influence of cleavage and polyadenylation on the evolution of genome architecture and provide resources for studying posttranscriptional gene regulation. Nine samples (10 sequencing runs) from various mixed and specific stages of wild-type Caenorhabditis elegans and glp-4 mutant adults.

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HIGH-THROUGHPUT IDENTIFICATION OF RNA LOCALIZATION ELEMENTS REVEALS A REGULATORY ROLE FOR A/G RICH SEQUENCES

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