A TARBP2 mutation in human cancer impairs microRNA processing and DICER1 function

Abstract

microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by targeting messenger RNA (mRNA) transcripts. Recently, a miRNA expression profile of human tumors has been characterized by an overall miRNA downregulation. Explanations for this observation include a failure of miRNA post-transcriptional regulation, transcriptional silencing associated with hypermethylation of CpG island promoters and miRNA transcriptional repression by oncogenic factors. Another possibility is that the enzymes and cofactors involved in miRNA processing pathways may themselves be targets of genetic disruption, further enhancing cellular transformation. However, no loss-of-function genetic alterations in the genes encoding these proteins have been reported. Here we have identified truncating mutations in TARBP2 (TAR RNA-binding protein 2), encoding an integral component of a DICER1-containing complex, in sporadic and hereditary carcinomas with microsatellite instability. The presence of TARBP2 frameshift mutations causes diminished TRBP protein expression and a defect in the processing of miRNAs. The reintroduction of TRBP in the deficient cells restores the efficient production of miRNAs and inhibits tumor growth. Most important, the TRBP impairment is associated with a destabilization of the DICER1 protein. These results provide, for a subset of human tumors, an explanation for the observed defects in the expression of mature miRNAs.

Figure 1

A mutant TARBP2 in human cancer. (a) Schematic representation of the TARBP2 gene, with the location of the (C)₇ and (C)₅ repeats, and electropherograms of TARBP2 wild-type (normal colon, RKO, HCT116 and SKUT1) and mutant (Co115 and SKUT1B) cells. (b) TRBP protein expression analyzed by protein blot was very low and diminished in the mutant Co115 and SKUT1B cells, respectively, but not in the other colon and endometrial cancer cell lines with a wild-type sequence. Quantitation values using densitometric software are shown. (c) Relative processing of endogenous precursor miRNAs in TARBP2 mutant (Co115) cell line was significantly reduced compared with TARBP2 wild-type (RKO and HCT116) cell lines (the results shown are the average of three independent studies *P < 0.001). Relative processing was defined as the ratio of mature to precursor miRNA. Error bars, s.e.m.

Figure 2

Transfection of wild-type TARBP2 rescues pre-miRNA processing capacity. (a) TRBP protein expression was restored after transfecting wild-type TARBP2 in Co115 cell line. Stable clones were selected treating cells with puromycin (left). As a control, the truncated form of TARBP2, due to the mutation in the (C)₅ repeat, was also transfected in a pCMV-Tag4b vector and tested using an antibody against Flag. Stable clones were selected using G418. (b) Co115 cell line transfected with wild-type TARBP2 presented a 3.6-fold increase in the capacity of processing the existing precursor miRNAs compared with cells transfected with empty vector. No changes were observed in cells transfected with the TARBP2 truncated form in comparison to cells transfected with empty vector. *P < 0.001. (c) RNA blot analysis confirmed a defect in the generation of mature miRNAs in TARBP2 mutant Co115 cells that was rescued by transfection of wild-type TARBP2. (d) Synthetic precursor molecules were transiently transfected in Co115 cells that stably expressed TARBP2 wild-type and truncated form, versus cell lines transfected with empty vector. Only in Co115 cells transfected with wild-type TARBP2 did we observe an increase in the capacity to process the transfected precursor molecules. **P < 0.005. (e) The use of a miRNA microarray platform showed an increase in the number of overexpressed mature miRNAs in the Co115 TARBP2-transfected cells, whereas transfection of the TARBP2 mutant form did not cause any miRNA overexpression. Error bars, s.e.m.

Figure 3

TARBP2 mutation impairs DICER1 protein. (a) DICER1 protein expression analyzed by protein blot was very low and diminished in the TARBP2 mutant Co115 and SKUT1B cell lines, respectively, but present in cancer cell lines with wild-type TARBP2 sequences. (b) DICER1 protein concentrations analyzed by protein blot were restored after transfection of wild-type TARBP2 in Co115 cell line. The transfection with the truncated form of TARBP2 did not restore DICER1 concentrations. (c) DICER1 protein concentrations were restored after transfecting DICER1 in Co115 cells. DICER1 transfection was unable to restore TRBP expression because these cells harbor the frameshift mutation in the TARBP2 gene. Stable clones were selected by treating cells with G418. (d) Co115 cells transfected with DICER1 show a mean 3.2-fold induction on the processing efficiency of precursor miRNAs in comparison cells transfected with empty vector. (e) Co115 cells transfected with DICER1 show a higher rate of processing synthetic precursor miRNA molecules in comparison to cells transfected with empty vector. Error bars, s.e.m.

Figure 4

Tumor suppressor features of the TRBP–DICER1 complex. (a) The MTT assay showed that mutant TARBP2 Co115 cells transfected with wild-type TARBP2 grew significantly slower compared to cells transfected with empty vector. Transfection with the TARBP2 mutant form did not affect growth. (b) The colony formation assay showed that mutant TARBP2 Co115 cells transfected with wild-type TARBP2 formed significantly less colonies in comparison to cells transfected with empty vector (*P < 0.001). Transfection with the TARBP2 mutant form did not affect colony formation. (c) Effect of TARBP2 transfection on the growth of Co115 cells in nude mice. Tumor volume was monitored over time, and the tumor was excised and weighed at 30 d. Note the large tumor on the left flank, corresponding to cells transfected with empty vector, and the reduced tumor on the opposite flank, corresponding to cells transfected with wild-type TARBP2. Transfection of mutant TARBP2 did not affect tumor growth. (d–f) Co115 cells transfected with DICER1 also showed reduced cell viability (d), diminished colony formation (e) and tumor growth inhibition in nude mice (f) in comparison to cells transfected with empty vector.