In vivo assembly of functional U7 snRNP requires RNA backbone flexibility within the Sm-binding site

Abstract

Most histone precursor mRNAs (pre-mRNAs) in metazoans are matured by 3′-end cleavage directed by the U7 small nuclear ribonucleoprotein (snRNP). RNA functional groups necessary for in vivo assembly and activity of the U7 snRNP were examined by nucleotide-analog interference mapping and mutagenesis using a chimeric mouse histone H4 pre-mRNA–U7 snRNA construct that is cleaved in cis in Xenopus laevis oocytes. Assembly of the unique U7 Sm protein core is rate limiting for processing in vivo and requires four conserved nucleotides within the U7 Sm-binding site, as well as the correct positioning and size of the U7 terminal stem-loop structure. To our surprise, pseudouridine substitution revealed a requirement for backbone flexibility at a particular position within the U7 Sm site, providing in vivo biochemical evidence that an unusual C2′-endo sugar conformation is necessary for assembly of the Sm ring.

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Figure 1: The histone pre-mRNA–U7 snRNA chimera.
Figure 2: U24Ψ interferes with U7 snRNP assembly in vivo.
Figure 3: The Y12 immunoprecipitation (IP) assay simultaneously assesses U7 Sm core assembly and processing efficiency of the chimera.
Figure 4: Spacing between the U7 terminal stem and the Sm site affects U7 snRNP assembly and processing.
Figure 5: Effects of single-base substitutions within the Sm-binding site on assembly and processing.

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Acknowledgements

We thank S. Strobel (Yale University) for generous gifts of 5′-O-(1-thio)-nucleoside analog triphosphates; R. Breaker, K. Tycowski and L. Weinstein-Szewczak for stimulating discussions; T. Steitz, K. Tycowski, S. Vasudevan and A. Alexandrov for critical reading of the manuscript; and A. Miccinello for secretarial help. This work was supported by US National Institutes of Health grant GM26154 to J.A.S. J.A.S. is an investigator of the Howard Hughes Medical Institute.

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Correspondence to Joan A Steitz.

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Supplementary information

Supplementary Fig. 1

Known and putative U7 snRNAs identified by BLAST search (PDF 29 kb)

Supplementary Fig. 2

Three-dimensional model of the hydrogen bond–mediated water bridge formed by Ψ in RNA (PDF 44 kb)

Supplementary Methods (PDF 38 kb)

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Kolev, N., Steitz, J. In vivo assembly of functional U7 snRNP requires RNA backbone flexibility within the Sm-binding site. Nat Struct Mol Biol 13, 347–353 (2006). https://doi.org/10.1038/nsmb1075

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