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Post-transcriptional processing generates a diversity of 5′-modified long and short RNAs

Abstract

The transcriptomes of eukaryotic cells are incredibly complex. Individual non-coding RNAs dwarf the number of protein-coding genes, and include classes that are well understood as well as classes for which the nature, extent and functional roles are obscure1. Deep sequencing of small RNAs (<200 nucleotides) from human HeLa and HepG2 cells revealed a remarkable breadth of species. These arose both from within annotated genes and from unannotated intergenic regions. Overall, small RNAs tended to align with CAGE (cap-analysis of gene expression) tags2, which mark the 5′ ends of capped, long RNA transcripts. Many small RNAs, including the previously described promoter-associated small RNAs3, appeared to possess cap structures. Members of an extensive class of both small RNAs and CAGE tags were distributed across internal exons of annotated protein coding and non-coding genes, sometimes crossing exon–exon junctions. Here we show that processing of mature mRNAs through an as yet unknown mechanism may generate complex populations of both long and short RNAs whose apparently capped 5′ ends coincide. Supplying synthetic promoter-associated small RNAs corresponding to the c-MYC transcriptional start site reduced MYC messenger RNA abundance. The studies presented here expand the catalogue of cellular small RNAs and demonstrate a biological impact for at least one class of non-canonical small RNAs.

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Figure 1: Genomic distribution of small RNAs.
Figure 2: Correlation of sRNAs and CAGE tags.
Figure 3: Correlation between CAGE tags, sRNAs and internal exons of annotated transcripts.
Figure 4: Regulation of gene expression by PASRs.
Figure 5: A proposed model for the metabolism of genic transcripts into a diversity of long and short RNAs.

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Gene Expression Omnibus

Data deposits

Sequences generated during this study have been deposited in GEO under accession number GSE14362.

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Acknowledgements

We thank L. Cardone, D. Rebolini, M. Kramer, and W. R. McCombie for Illumina sequencing. We wish to thank J. Brosius, J. Schmitz and T. Rozhdestvensky for their help with the small RNA cloning protocol and J. Dumais for technical assistance. K.F.-T. was in part supported by the Schering Foundation. This work was supported in part by grants from the NIH and was performed as part of the ENCODE consortium (G.J.H. and T.R.G.). G.J.H is an investigator of the Howard Hughes Medical Institute.

Author Contributions K.F.-T. and P.K. performed experiments in collaboration with E.D., V.S., R.D. and A.T.W. P.K., S.F., R.S. and G.A. performed data analysis. G.J.H. and T.R.G. planned experiments and wrote the paper.

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Correspondence to Gregory J. Hannon or Thomas R. Gingeras.

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R.D. is an employee of Affymetrix, which manufactures the tiling arrays used in the study.

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This file contains Supplementary Methods, Supplementary References, Supplementary Figures S1-S3 with Legends and Supplementary Tables S1-S2 (PDF 1208 kb)

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Affymetrix/Cold Spring Harbor Laboratory ENCODE Transcriptome Project. Post-transcriptional processing generates a diversity of 5′-modified long and short RNAs. Nature 457, 1028–1032 (2009). https://doi.org/10.1038/nature07759

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