Gene Architecture and Sequence Composition Underpin Selective Dependency of Nuclear Export of Long RNAs on NXF1 and the TREX Complex.

The core components of the nuclear RNA export pathway are thought to be required for export of virtually all polyadenylated RNAs. Here, we depleted different proteins that act in nuclear export in human cells and quantified the transcriptome-wide consequences on RNA localization. Different genes exhibited substantially variable sensitivities, with depletion of NXF1 and TREX components causing some transcripts to become strongly retained in the nucleus while others were not affected. Specifically, NXF1 is preferentially required for export of single- or few-exon transcripts with long exons or high A/U content, whereas depletion of TREX complex components preferentially affects spliced and G/C-rich transcripts. Using massively parallel reporter assays, we identified short sequence elements that render transcripts dependent on NXF1 for their export and identified synergistic effects of splicing and NXF1. These results revise the current model of how nuclear export shapes the distribution of RNA within human cells.

Massively parallel identification of mRNA localization elements in primary cortical neurons.

Cells adopt highly polarized shapes and form distinct subcellular compartments in many cases due to the localization of many mRNAs to specific areas, where they are translated into proteins with local functions. This mRNA localization is mediated by specific cis-regulatory elements in mRNAs, commonly called 'zipcodes'. Although there are hundreds of localized mRNAs, only a few zipcodes have been characterized. Here we describe a novel neuronal zipcode identification protocol (N-zip) that can identify zipcodes across hundreds of 3' untranslated regions. This approach combines a method of separating the principal subcellular compartments of neurons-cell bodies and neurites-with a massively parallel reporter assay. N-zip identifies the let-7 binding site and (AU)n motif as de novo zipcodes in mouse primary cortical neurons. Our analysis also provides, to our knowledge, the first demonstration of an miRNA affecting mRNA localization and suggests a strategy for detecting many more zipcodes.

Context-specific effects of sequence elements on subcellular localization of linear and circular RNAs.

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.