ABSTRACT
Stimulated cells and cancer cells have widespread shortening of mRNA 3'-untranslated regions (3'UTRs) and switches to shorter mRNA isoforms due to usage of more proximal polyadenylation signals (PASs) in introns and last exons. U1 snRNP (U1), vertebrates' most abundant non-coding (spliceosomal) small nuclear RNA, silences proximal PASs and its inhibition with antisense morpholino oligonucleotides (U1 AMO) triggers widespread premature transcription termination and mRNA shortening. Here we show that low U1 AMO doses increase cancer cells' migration and invasion in vitro by up to 500%, whereas U1 over-expression has the opposite effect. In addition to 3'UTR length, numerous transcriptome changes that could contribute to this phenotype are observed, including alternative splicing, and mRNA expression levels of proto-oncogenes and tumor suppressors. These findings reveal an unexpected role for U1 homeostasis (available U1 relative to transcription) in oncogenic and activated cell states, and suggest U1 as a potential target for their modulation.
Subject(s)
Cell Movement , Neoplasms/metabolism , Ribonucleoprotein, U1 Small Nuclear/metabolism , Cell Line, Tumor , Humans , Neoplasm Invasiveness , Neoplasms/genetics , Neoplasms/pathology , Neoplasms/physiopathology , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/metabolism , RNA Splicing , RNA, Messenger/metabolism , Ribonucleoprotein, U1 Small Nuclear/geneticsABSTRACT
Full-length transcription in the majority of human genes depends on U1 snRNP (U1) to co-transcriptionally suppress transcription-terminating premature 3' end cleavage and polyadenylation (PCPA) from cryptic polyadenylation signals (PASs) in introns. However, the mechanism of this U1 activity, termed telescripting, is unknown. Here, we captured a complex, comprising U1 and CPA factors (U1-CPAFs), that binds intronic PASs and suppresses PCPA. U1-CPAFs are distinct from U1-spliceosomal complexes; they include CPA's three main subunits, CFIm, CPSF, and CstF; lack essential splicing factors; and associate with transcription elongation and mRNA export complexes. Telescripting requires U1:pre-mRNA base-pairing, which can be disrupted by U1 antisense oligonucleotide (U1 AMO), triggering PCPA. U1 AMO remodels U1-CPAFs, revealing changes, including recruitment of CPA-stimulating factors, that explain U1-CPAFs' switch from repressive to activated states. Our findings outline this U1 telescripting mechanism and demonstrate U1's unique role as central regulator of pre-mRNA processing and transcription.
Subject(s)
Cell Nucleus/metabolism , Cleavage And Polyadenylation Specificity Factor/metabolism , RNA Cleavage , RNA Precursors/biosynthesis , RNA, Messenger/biosynthesis , Ribonucleoprotein, U1 Small Nuclear/metabolism , Transcription, Genetic , 3' Untranslated Regions , Active Transport, Cell Nucleus , Binding Sites , Cell Nucleus/genetics , Cleavage And Polyadenylation Specificity Factor/genetics , Cleavage Stimulation Factor/genetics , Cleavage Stimulation Factor/metabolism , HeLa Cells , Humans , Multiprotein Complexes , Poly A/metabolism , Protein Binding , RNA Precursors/genetics , RNA, Messenger/genetics , Ribonucleoprotein, U1 Small Nuclear/geneticsABSTRACT
Recent observations showed that nascent RNA polymerase II transcripts, pre-mRNAs, and noncoding RNAs are highly susceptible to premature 3'-end cleavage and polyadenylation (PCPA) from numerous intronic cryptic polyadenylation signals (PASs). The importance of this in gene regulation was not previously appreciated as PASs, despite their prevalence, were thought to be active in terminal exons at gene ends. Unexpectedly, antisense oligonucleotide interference with U1 snRNA base-pairing to 5' splice sites, which is necessary for U1 snRNP's (U1) function in splicing, caused widespread PCPA in metazoans. This uncovered U1's PCPA suppression activity, termed telescripting, as crucial for full-length transcription in thousands of vertebrate genes, providing a general role in transcription elongation control. Progressive intron-size expansion in metazoan evolution greatly increased PCPA vulnerability and dependence on U1 telescripting. We describe how these observations unfolded and discuss U1 telescripting's role in shaping the transcriptome.
Subject(s)
Gene Expression Regulation/physiology , RNA, Small Nuclear/metabolism , Ribonucleoproteins, Small Nuclear/metabolism , Transcription Termination, Genetic , Animals , Humans , Introns , RNA, Small Nuclear/geneticsABSTRACT
U1 snRNP (U1) functions in splicing introns and telescripting, which suppresses premature cleavage and polyadenylation (PCPA). Using U1 inhibition in human cells, we show that U1 telescripting is selectively required for sustaining long-distance transcription elongation in introns of large genes (median 39 kb). Evidence of widespread PCPA in the same locations in normal tissues reveals that large genes incur natural transcription attrition. Underscoring the importance of U1 telescripting as a gene-size-based mRNA-regulation mechanism, small genes were not sensitive to PCPA, and the spliced-mRNA productivity of â¼1,000 small genes (median 6.8 kb) increased upon U1 inhibition. Notably, these small, upregulated genes were enriched in functions related to acute stimuli and cell-survival response, whereas genes subject to PCPA were enriched in cell-cycle progression and developmental functions. This gene size-function polarization increased in metazoan evolution by enormous intron expansion. We propose that telescripting adds an overarching layer of regulation to size-function-stratified genomes, leveraged by selective intron expansion to rapidly shift gene expression priorities.
