Maya Ron, Igor Ulitsky
Long RNAs vary extensively in their post-transcriptional fates, and this variation is attributed in part to short sequence elements. We used massively parallel RNA assays to study how sequences derived from noncoding RNAs influence the subcellular localization and stability of circular and linear RNAs, including spliced and unspliced forms. We find that the effects of sequence elements strongly depend on the host RNA context, with limited overlap between sequences that drive nuclear enrichment of linear and circular RNAs. Binding of specific RNA binding proteins underpins some of these differences—SRSF1 binding leads to nuclear enrichment of circular RNAs; SAFB binding is associated with nuclear enrichment of predominantly unspliced linear RNAs; and IGF2BP1 promotes export of linear spliced RNA molecules. The post-transcriptional fate of long RNAs is thus dictated by combinatorial contributions of specific sequence elements, of splicing, and of the presence of the terminal features unique to linear RNAs.
Maya Ron, Igor Ulitsky. Context-specific effects of sequence elements on subcellular localization of linear and circular RNAs. 2022-05-05. https://doi.org/10.1038/s41467-022-30183-0
CircLibA and NucLibA sequence tiles were cloned into WT beta-globin (spliced), beta-globin-Delta introns (unspliced), circPVT1, and scrambled circPVT1 reporter backbones and transfected into MCF-7 cells. Nuclear, cytoplasmic, whole-cell, and plasmid-input measurements were used to report context-specific localization and expression effects.
MCF-7 cells were treated with siRNA pools targeting SRSF1, SAFB, IGF2BP1, or IGF2BP2, followed by transfection of the reporter libraries and nuclear/cytoplasmic fractionation. The table reports the authors' median control and knockdown localization/expression scores and knockdown-minus-control localization shifts for spliced, unspliced, and circular reporter contexts.
The same sequence-tile reporters were transfected into MCF-7 cells and transcription was inhibited with actinomycin D. Expression was measured at 2, 4, and 8 h, normalized to spiked Renilla luciferase controls, and used to estimate tile half-lives in spliced, unspliced, and circular reporter contexts.
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