hnRNP H1 and intronic G runs in the splicing control of the human rpL3 gene.
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.
Russo
Annapina
A
Dipartimento di Biochimica e Biotecnologie Mediche, Università Federico II, Napoli 80131, Italy.
Siciliano
Gabriella
G
Catillo
Morena
M
Giangrande
Chiara
C
Amoresano
Angela
A
Pucci
Pietro
P
Pietropaolo
Concetta
C
Russo
Giulia
G
D017398:Alternative Splicing; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D006367:HeLa Cells; D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007438:Introns; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D022821:RNA Splice Sites; D034741:RNA, Small Interfering / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000090822:Ribosomal Protein L3; D012269:Ribosomal Proteins / Q000235:genetics* / Q000378:metabolism; D014163:Transfer, Psychology
D017931:DNA Primers; D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H; D012322:RNA Precursors; D022821:RNA Splice Sites; D034741:RNA, Small Interfering; C000716547:RPL3 protein, human; D011993:Recombinant Fusion Proteins; D000090822:Ribosomal Protein L3; D012269:Ribosomal Proteins