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A U1 snRNP–specific assembly pathway reveals the SMN complex as a versatile hub for RNP exchange

Nature Structural & Molecular Biology volume 23, pages 225–230 (2016)Cite this article

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Abstract

Despite equal snRNP stoichiometry in spliceosomes, U1 snRNP (U1) is typically the most abundant vertebrate snRNP. Mechanisms regulating U1 overabundance and snRNP repertoire are unknown. In Sm-core assembly, a key snRNP-biogenesis step mediated by the SMN complex, the snRNA-specific RNA-binding protein (RBP) Gemin5 delivers pre-snRNAs, which join SMN–Gemin2–recruited Sm proteins. We show that the human U1-specific RBP U1-70K can bridge pre-U1 to SMN–Gemin2–Sm, in a Gemin5-independent manner, thus establishing an additional and U1-exclusive Sm core–assembly pathway. U1-70K hijacks SMN–Gemin2–Sm, enhancing Sm-core assembly on U1s and inhibiting that on other snRNAs, thereby promoting U1 overabundance and regulating snRNP repertoire. SMN–Gemin2's ability to facilitate transactions between different RBPs and RNAs explains its multi-RBP valency and the myriad transcriptome perturbations associated with SMN deficiency in neurodegenerative spinal muscular atrophy. We propose that SMN–Gemin2 is a versatile hub for RNP exchange that functions broadly in RNA metabolism.

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Figure 1: The U1 snRNP–specific stem-loop 1–binding protein U1-70K bridges pre-U1 or U1 snRNA to the SMN complex independently of Gemin5.
Figure 2: U1-70K enhances Sm-core assembly on U1 and inhibits that on other snRNAs in vitro, and regulates the snRNP repertoire in cells.
Figure 3: U1-70K bridges pre-U1 snRNA to SMN and mediates preferential Sm-core assembly on U1 snRNA rather than on other snRNAs.
Figure 4: SMN–Gemin2 cooperates with U1-70K in Sm-protein recruitment.
Figure 5: Schematic representation of SMN–Gemin2's function as a versatile hub for RNP exchange.

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Acknowledgements

We thank members of our laboratory for helpful discussions and comments on the manuscript. This work was supported by the Association Française Contre les Myopathies (AFM) and by the US National Institutes of Health (R01 GM112923 to G.D.) G.D. is supported as an Investigator of the Howard Hughes Medical Institute.

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  1. Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA

    Byung Ran So, Lili Wan, Zhenxi Zhang, Pilong Li, Eric Babiash, Jingqi Duan, Ihab Younis & Gideon Dreyfuss

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Contributions

B.R.S., L.W. and Z.Z. designed and performed experiments. B.R.S., L.W., Z.Z., P.L., E.B., J.D. and I.Y. contributed to data analysis. G.D. is responsible for the project's planning and experimental design. All authors contributed to writing the paper.

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Correspondence to Gideon Dreyfuss.

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Integrated supplementary information

Supplementary Figure 1 Western blot analysis of the Gemin2-knockdown cell extracts used for in vitro Sm-core assembly.

Input cytoplasmic extracts from HeLa cells, transfected with control siRNA and siRNA targeting against Gemin2, are shown. Percent of residual Gemin2 protein after knockdown compared to control (100%) is indicated.

Supplementary Figure 2 Western blot analysis of the U1-70K-, SMN- or Gemin5-knockdown cell extracts used for snRNP-level measurements.

The SMN complex, U1-70K and Sm proteins in control, U1-70K, SMN or Gemin5 knockdown HeLa cells are shown. FXR1 was used as a loading control. Residual knockdown proteins for each knockdown compared to control (100%) are indicated.

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Supplementary Text and Figures

Supplementary Figures 1 and 2 (PDF 8152 kb)

Supplementary Data Set 1

Uncropped blots and gels with size marker indications (PDF 8111 kb)

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So, B., Wan, L., Zhang, Z. et al. A U1 snRNP–specific assembly pathway reveals the SMN complex as a versatile hub for RNP exchange. Nat Struct Mol Biol 23, 225–230 (2016). https://doi.org/10.1038/nsmb.3167

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