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U1 snRNP protects pre-mRNAs from premature cleavage and polyadenylation

Abstract

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

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Figure 1: U1 AMO binds to the 5′ sequence of U1 snRNA and inhibits its splicing activity.
Figure 2: Genomic tiling arrays identify unspliced pre-mRNAs after U1 AMO and SSA treatment.
Figure 3: Premature termination in introns of pre-mRNAs in U1-AMO-transfected cells.
Figure 4: Prematurely terminated pre-mRNAs are polyadenylated from cryptic PASs in introns.
Figure 5: U1 snRNP suppression of premature cleavage and polyadenylation from a nearby cryptic PAS is splicing independent and requires base pairing.

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Gene Expression Omnibus

Data deposits

The tiling array data have been submitted to the GEO database under the accession number GSE24179.

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Acknowledgements

We are grateful to the members of our laboratory, especially J. Yong and J. Bachorik, for discussions and comments on this manuscript. We thank M. Yoshida for providing spliceostatin A. We also thank D. A. Baldwin and H. Rodriguez at the Microarray Core Facility at the University of Pennsylvania School of Medicine for help with the tiling array. This work was supported by the Association Française Contre les Myopathies (AFM). G.D. is an Investigator of the Howard Hughes Medical Institute.

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D.K., M.G.B., I.Y., M.K., L.N.S. and L.W. designed and performed experiments and contributed to data analysis. G.D. is responsible for the project planning and experimental design. All authors contributed to writing the paper.

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Correspondence to Gideon Dreyfuss.

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The authors declare no competing financial interests.

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Kaida, D., Berg, M., Younis, I. et al. U1 snRNP protects pre-mRNAs from premature cleavage and polyadenylation. Nature 468, 664–668 (2010). https://doi.org/10.1038/nature09479

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