SummaryIn higher eukaryotes, U1 snRNP forms spliceosomes in equal stoichiometry with U2, U4, U5 and U6, however its abundance far exceeds that of the other snRNPs. Here, we used antisense morpholino oligonucleotide (AMO) to U1 snRNA for functional U1 snRNP knockdown in HeLa cells and identified accumulated unspliced pre-mRNAs by genomic tiling microarrays. Remarkably, in addition to inhibiting splicing, U1 snRNP knockdown caused premature cleavage and polyadenylation (PCPA) in numerous pre-mRNAs at cryptic polyadenylation signals (PASs), frequently in introns near (< 5 kb) the start of the transcript. This did not occur when splicing was inhibited with U2 snRNA AMO or the U2 snRNP inactivating drug, spliceostatin A, unless U1 AMO was also included. We further show that U1 snRNA-pre-mRNA base pairing was required to suppress PCPA from nearby cryptic PASs located in introns. These findings reveal a critical splicing-independent function for U1 snRNP in protecting the transcriptome, which we propose explains its overabundance.

U1 snRNP (U1), in addition to its splicing role, protects pre-mRNAs from drastic premature termination by cleavage and polyadenylation (PCPA) at cryptic polyadenylation signals (PASs) in introns. Here, a high-throughput sequencing strategy of differentially expressed transcripts (HIDE-seq) mapped PCPA sites genome wide in divergent organisms. Surprisingly, whereas U1 depletion terminated most nascent gene transcripts within ~1 kb, moderate functional U1 level decreases, insufficient to inhibit splicing, dose-dependently shifted PCPA downstream and elicited mRNA 3' UTR shortening and proximal 3' exon switching characteristic of activated immune and neuronal cells, stem cells, and cancer. Activated neurons' signature mRNA shortening could be recapitulated by U1 decrease and antagonized by U1 overexpression. Importantly, we show that rapid and transient transcriptional upregulation inherent to neuronal activation physiology creates U1 shortage relative to pre-mRNAs. Additional experiments suggest cotranscriptional PCPA counteracted by U1 association with nascent transcripts, a process we term telescripting, ensuring transcriptome integrity and regulating mRNA length.

Mining the genome sequence of Burkholderia thailandensis MSMB43 revealed a cryptic biosynthetic gene cluster resembling that of FR901464 (4), a prototype spliceosome inhibitor produced by Pseudomonas sp. No. 2663. Transcriptional analysis revealed a cultivation condition in which a regulatory gene of the cryptic gene cluster is adequately expressed. Consequently, three new compounds, named thailanstatins A (1), B (2) and C (3), were isolated from the fermentation broth of B. thailandensis MSMB43. Thailanstatins are proposed to be biosynthesized by a hybrid polyketide synthase-nonribosomal peptide synthetase pathway. They differ from 4 by lacking an unstable hydroxyl group and by having an extra carboxyl moiety; those differences endow thailanstatins with a significantly greater stability than 4 as tested in phosphate buffer at pH 7.4. In vitro assays showed that thailanstatins inhibit pre-mRNA splicing as potently as 4, with half-maximal inhibitory concentrations in the single to sub µM range. Cell culture assays indicated that thailanstatins also possess potent antiproliferative activities in representative human cancer cell lines, with half-maximal growth inhibitory concentrations in the single nM range. This work provides new chemical entities for research and development, and new structure-activity information for chemical optimization of related spliceosome inhibitors.