Nuclear retention of unspliced pre-mRNAs by mutant DHX16/hPRP2, a spliceosomal DEAH-box protein.
Defective or imbalanced expression of spliceosomal factors has been linked to human disease; however, how a defective spliceosome affects intron-containing gene transcripts in human cells is largely unknown. DEAH-box protein DHX16 is a human orthologue of Saccharomyces cerevisiae spliceosomal protein Prp2, an RNA-dependent ATPase that activates the spliceosome before the first catalytic step of splicing. Yeast prp2 mutants accumulate unspliced RNAs from the vast majority of intron-containing genes. Here we used a genomic tiling microarray to screen transcripts from four chromosomes in human cells expressing a dominant negative DHX16 mutant and identified a number of gene transcripts that retained their introns. The mutant protein also affected gene transcripts that are sensitive to pladienolide, an SF3b inhibitor. The unspliced RNAs were retained in the nucleus, and block of nonsense-mediated decay did not affect their accumulation. Thus, a perturbation of human PRP2/DHX16 results in accumulation of unspliced transcripts, similar to the outcome in yeast prp2 mutants. The results further suggest that mutant DHX16/hPRP2 causes a defective spliceosome to retain unspliced gene transcripts in the nuclei of human cells.
Gencheva
Marieta
M
Department of Molecular and Cellular Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Lin
Ting-Yu
TY
Wu
Xiwei
X
Yang
Lixin
L
Richard
Caroline
C
Jones
Matthew
M
Lin
Shwu-Bin
SB
Lin
Ren-Jang
RJ
D019943:Amino Acid Substitution; D015152:Blotting, Northern; D015153:Blotting, Western; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D057809:HEK293 Cells; D006801:Humans; D007438:Introns / Q000235:genetics; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D012313:RNA / Q000235:genetics / Q000378:metabolism; D020365:RNA Helicases / Q000235:genetics* / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics*; D012326:RNA Splicing; D020871:RNA Stability; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017381:Spliceosomes / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D012322:RNA Precursors; D012313:RNA; C555647:DHX16 protein, human; D020365:RNA Helicases