HnRNP L binds a cis-acting RNA sequence element that enables intron-dependent gene expression.
Most pre-mRNAs require an intron for efficient processing in higher eukaryotes. To test the hypothesis that intron-independent gene expression involves positive, cis-acting RNA sequence elements, we constructed chimeric genes in which various regions of the naturally intronless HSV-TK gene were inserted into an intronless variant of the highly intron-dependent human beta-globin gene. Using a transient transfection assay, we identified a 119-nucleotide sequence element contained within the transcribed region of the HSV-TK gene that enables efficient cytoplasmic accumulation of globin RNA in the absence of splicing. RNA UV-cross-linking assays indicated that a 68-kD protein present in nuclear extracts of HeLa and COS cells specifically binds to this HSV-TK sequence element. This 68-kD protein was found to cross-react with an antiserum specific to hnRNP L. Recombinant hnRNP L was shown to bind with high sequence specificity to this RNA sequence element. Analysis of substitution mutants in this element indicated that binding of hnRNP L correlates with accumulation of the RNA in the cytoplasm. Thus, we conclude that (1) hnRNP L binds in a sequence-specific manner to this RNA sequence element that enables intron-independent gene expression, and (2) intron-independent pre-mRNA processing and transport involves sequence-specific RNA-protein interactions between cis-acting RNA sequence elements and proteins such as hnRNP L. This sequence element may be of general use for the efficient expression of cDNA versions of intron-dependent genes.
Liu
X
X
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706-1559, USA.
Mertz
J E
JE
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002678:Chimera / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004742:Enhancer Elements, Genetic; D015870:Gene Expression; D005914:Globins / Q000235:genetics; D034664:Heterogeneous-Nuclear Ribonucleoprotein L; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D005810:Multigene Family; D009154:Mutation; D012313:RNA / Q000235:genetics* / Q000378:metabolism*; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing / Q000235:genetics; D012261:Ribonucleoproteins / Q000378:metabolism*; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics
D017931:DNA Primers; D018076:DNA, Complementary; D034664:Heterogeneous-Nuclear Ribonucleoprotein L; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D012322:RNA Precursors; D012261:Ribonucleoproteins; D012313:RNA; D005914:Globins; D013937:Thymidine Kinase