Yoav Lubelsky, Igor Ulitsky
Long noncoding RNAs (lncRNAs) are emerging as key parts of multiple cellular pathways, but their modes of action and how these are dictated by sequence remain unclear. lncRNAs tend to be enriched in the nuclear fraction, whereas most mRNAs are overtly cytoplasmic, although several studies have found that hundreds of mRNAs in various cell types are retained in the nucleus. It is thus conceivable that some mechanisms that promote nuclear enrichment are shared between lncRNAs and mRNAs. Here, to identify elements in lncRNAs and mRNAs that can force nuclear localization, we screened libraries of short fragments tiled across nuclear RNAs, which were cloned into the untranslated regions of an efficiently exported mRNA. The screen identified a short sequence derived from Alu elements and bound by HNRNPK that increased nuclear accumulation. Binding of HNRNPK to C-rich motifs outside Alu elements is also associated with nuclear enrichment in both lncRNAs and mRNAs, and this mechanism is conserved across species. Our results thus identify a pathway for regulation of RNA accumulation and subcellular localization that has been co-opted to regulate the fate of transcripts with integrated Alu elements.
Yoav Lubelsky, Igor Ulitsky. Sequences enriched in Alu repeats drive nuclear localization of long RNAs in human cells. 2018-01-24. https://doi.org/10.1038/nature25757
A barcoded 109-nt tile library from human lncRNAs, mouse liver nuclear-enriched mRNA 3' UTRs, and MALAT1 homologs was cloned into the 3' or 5' UTR of AcGFP in episomal plasmids. MCF7 cells were transfected in triplicate, and whole-cell, nuclear, cytoplasmic, and input libraries were sequenced to quantify localization and expression effects.
A second barcoded 109-nt library was cloned into the 3' UTR of AcGFP and included transcript tiles, wild-type SIRLOIN-containing elements, and systematic sequence variants of JPX#9 and PVT1#22. MCF7 cells were transfected in two replicates and nuclear/cytoplasmic localization plus whole-cell-extract/input abundance were measured by sequencing.
The NucLibB AcGFP 3' UTR library was transfected into MCF7 cells after HNRNPK siRNA or non-targeting siRNA treatment. The released source data report per-element knockdown-minus-control differences in nuclear/cytoplasmic, whole-cell-extract/input, nuclear/input, and cytoplasmic/input log2 ratios.
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