Alterations in Gemin5 expression contribute to alternative mRNA splicing patterns and tumor cell motility.
The role of Gemin5 in alternative mRNA splicing, tumor cell motility, and proteomic instability was investigated. Isotope Capture Affinity Tag proteomic analysis was conducted on MDA-MB-435 tumor cells transfected with either a control vector (C-100) or the Nm23-H1 metastasis suppressor (H1-177). Ingenuity pathway analysis revealed that RNA posttranscriptional processing was the most prominent class of differentially expressed proteins. Within this category, overexpression of Acinus1, Poly(a) binding protein, HNRPA2B1, Bop1, and Gemin5 was confirmed in less metastatic H1-177 cells. Overexpression of the latter four proteins was also observed in the lower metastatic antisense Ezrin transfectant of a murine osteosarcoma model system, confirming the general relevance of the trends. Gemin5, a component of the spliceosomal complex, was chosen for further study. Analysis of global mRNA splicing by SpliceArray chips revealed that 16 genes were differentially spliced in C-100 compared with H1-177 cells; transient transfection of gemin5 into C-100 cells restored the splice pattern to that of H1-177 cells. Alternative splicing patterns for the engulfment and cell motility 1 and thrombospondin 4 genes were confirmed by semiquantitative reverse transcription-PCR. Gemin5 overexpression coordinately reduced C-100 cell motility by 50%, and siRNA-mediated reduction of Gemin5 expression increased the motility of H1-177 cells by 2-fold (P < 0.004). The data provide the first demonstration that alterations in the expression of a spliceosome protein can effect both specific splicing events and tumor cell motility. The data also show that changes in mRNA splicing patterns accompany metastatic progression, which may contribute to proteome instability.
Lee
Jong Heun
JH
Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA.
Horak
Christine E
CE
Khanna
Chand
C
Meng
Zhaojing
Z
Yu
Li Rong
LR
Veenstra
Timothy D
TD
Steeg
Patricia S
PS
D017398:Alternative Splicing; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics*; D006801:Humans; D054778:NM23 Nucleoside Diphosphate Kinases / Q000096:biosynthesis / Q000235:genetics; D009362:Neoplasm Metastasis; D020033:Protein Isoforms; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear / Q000096:biosynthesis* / Q000235:genetics; D055532:SMN Complex Proteins; D014162:Transfection
C449808:GEMIN5 protein, human; D054778:NM23 Nucleoside Diphosphate Kinases; D020033:Protein Isoforms; D012333:RNA, Messenger; D017411:Ribonucleoproteins, Small Nuclear; D055532:SMN Complex Proteins; C517938:NME1 protein, human