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Splice site strength–dependent activity and genetic buffering by poly-G runs

Nature Structural & Molecular Biology volume 16, pages 1094–1100 (2009)Cite this article

Abstract

Pre-mRNA splicing is regulated through the combinatorial activity of RNA motifs, including splice sites and splicing regulatory elements. Here we show that the activity of the G-run (polyguanine sequence) class of splicing enhancer elements is ∼4-fold higher when adjacent to intermediate strength 5′ splice sites (ss) than when adjacent to weak 5′ ss, and ∼1.3-fold higher relative to strong 5′ ss. We observed this dependence on 5′ ss strength in both splicing reporters and in global microarray and mRNA-Seq analyses of splicing changes following RNA interference against heterogeneous nuclear ribonucleoprotein (hnRNP) H, which cross-linked to G-runs adjacent to many regulated exons. An exon's responsiveness to changes in hnRNP H levels therefore depends in a complex way on G-run abundance and 5′ ss strength. This pattern of activity enables G-runs and hnRNP H to buffer the effects of 5′ ss mutations, augmenting both the frequency of 5′ ss polymorphism and the evolution of new splicing patterns. Certain other splicing factors may function similarly.

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Figure 1: Abundance, conservation and 5′ ss strength–dependent activity of G-run ISEs.
Figure 2: hnRNP H knockdown and mRNA-Seq analysis of G-run activity.
Figure 3: Genetic buffering by G-runs.
Figure 4: Sequence conservation flanking exons is dependent on 5′ ss strength.

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Acknowledgements

We thank F. Allain and K. Lynch for helpful discussions, M. McNally, C. Nielsen, R. Sandberg, P. A. Sharp and members of the Burge laboratory for helpful comments on this manuscript and G.P. Schroth and his research group for high-throughput cDNA sequencing. This work was supported by postdoctoral fellowships from the American Heart Association (X.X.) and the Human Frontiers Science Program (R.N.), by a training grant from the US National Institutes of Health (NIH) (E.T.W.), by US National Science Foundation (NSF) equipment grant DBI-0821391 and by grants from the NIH (C.B.B.).

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Author notes

  1. Xinshu Xiao & Zefeng Wang

    Present address: Present addresses: Department of Physiological Science and the Molecular Biology Institute, University of California, Los Angeles, California, USA (X.X.); Department of Pharmacology, University of North Carolina at Chapel Hill, North Carolina, USA (Z.W.).,

Authors and Affiliations

  1. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Xinshu Xiao, Zefeng Wang, Minyoung Jang, Razvan Nutiu, Eric T Wang & Christopher B Burge

  2. Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Eric T Wang

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  1. Xinshu Xiao
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Contributions

X.X. designed and executed experiments and computational analyses, developed the evolutionary model, analyzed data and prepared the figures; Z.W. designed and conducted splicing reporter experiments and analyzed data; M.J. conducted cloning and splicing reporter experiments; R.N. designed and executed the RNAi and qRT-PCR experiments; E.T.W. designed and executed the CLIP-Seq experiments and related computational analyses; C.B.B. contributed to design of experiments and computational analyses and interpretation of data and wrote the paper with input from the other authors.

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Correspondence to Christopher B Burge.

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Xiao, X., Wang, Z., Jang, M. et al. Splice site strength–dependent activity and genetic buffering by poly-G runs. Nat Struct Mol Biol 16, 1094–1100 (2009). https://doi.org/10.1038/nsmb.1661

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