hnRNP H1 and intronic G runs in the splicing control of the human rpL3 gene

Biochim Biophys Acta. May-Jun 2010;1799(5-6):419-28. doi: 10.1016/j.bbagrm.2010.01.008. Epub 2010 Jan 25.

Abstract

By generating mRNA containing a premature termination codon (PTC), alternative splicing (AS) can quantitatively regulate the expression of genes that are degraded by nonsense-mediated mRNA decay (NMD). We previously demonstrated that AS-induced retention of part of intron 3 of rpL3 pre-mRNA produces an mRNA isoform that contains a PTC and is targeted for decay by NMD. We also demonstrated that overexpression of rpL3 downregulates canonical splicing and upregulates the alternative splicing of its pre-mRNA. We are currently investigating the molecular mechanism underlying rpL3 autoregulation. Here we report that the heterogeneous nuclear ribonucleoprotein (hnRNP) H1 is a transacting factor able to interact in vitro and in vivo with rpL3 and with intron 3 of the rpL3 gene. We investigated the role played by hnRNP H1 in the regulation of splicing of rpL3 pre-mRNA by manipulating its expression level. Depletion of hnRNP H1 reduced the level of the PTC-containing mRNA isoform, whereas its overexpression favored the selection of the cryptic 3' splice site of intron 3. We also identified and characterized the cis-acting regulatory elements involved in hnRNP H1-mediated regulation of splicing. RNA electromobility shift assay demonstrated that hnRNP H1 specifically recognizes and binds directly to the intron 3 region that contains seven copies of G-rich elements. Site-directed mutagenesis analysis and in vivo studies showed that the G3 and G6 elements are required for hnRNP H1-mediated regulation of rpL3 pre-mRNA splicing. We propose a working model in which rpL3 recruits hnRNP H1 and, through cooperation with other splicing factors, promotes selection of the alternative splice site.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alternative Splicing
  • Base Sequence
  • Binding Sites / genetics
  • Cell Line
  • DNA Primers / genetics
  • HeLa Cells
  • Heterogeneous-Nuclear Ribonucleoprotein Group F-H / antagonists & inhibitors
  • Heterogeneous-Nuclear Ribonucleoprotein Group F-H / genetics
  • Heterogeneous-Nuclear Ribonucleoprotein Group F-H / metabolism*
  • Humans
  • In Vitro Techniques
  • Introns
  • Models, Biological
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • RNA Precursors / genetics
  • RNA Precursors / metabolism
  • RNA Splice Sites
  • RNA, Small Interfering / genetics
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Ribosomal Proteins / genetics*
  • Ribosomal Proteins / metabolism
  • Transfer, Psychology

Substances

  • DNA Primers
  • Heterogeneous-Nuclear Ribonucleoprotein Group F-H
  • RNA Precursors
  • RNA Splice Sites
  • RNA, Small Interfering
  • Recombinant Fusion Proteins
  • Ribosomal Proteins
  • ribosomal protein L3