PMC

(A) WT melastatin mini-gene was mutated at indicated positions to generate constructs that lack the consensus 5′SS, the consensus 3′SS, three predicted branch points (3BP), six predicted branch points (6BP), two pyrimidines in the polypyrimidine tract (2Y), or four pyrimidines in the polypyrimidine tract (4Y). (B) Microprocessing of miR-211 is reduced by mutation of the 5′SS of the melastatin mini-gene, but is not affected by mutations in sequences involved in events downstream of 5′SS recognition. Either empty vector (EV) or vectors containing WT or mutant melastatin mini-genes were transfected into HeLa cells, and the efficiency of splicing was assessed by qRT-PCR and the efficiency of intronic miR-211 processing was assessed by Northern blotting. (C) Binding of Drosha to the miR-211-containing intron 6 of melastatin is reduced after depletion of splicing factors involved in U1 (SNRNP70+PRP8), but not U2 (U2AF65+PRP8) or U4/5/6 (PRP4+PRP8) snRNP function. HeLa cells were transfected with either Scr or splicing-factor-specific siRNAs, and then were co-transfected with vectors expressing WT melastatin mini-gene and either empty (EV) or FLAG-tagged Drosha. Anti-FLAG-Drosha immunoprecipitates and inputs were assayed for spliced and unspliced melastatin mini-gene transcripts by qRT-PCR (p<0.05). The efficiency of FLAG-Drosha immunoprecipitation was assessed by Western blotting. (D) Mutation of 5′SS of the melastatin mini-gene reduces Drosha binding to miR-211-hosting intron 6. Empty (EV) or FLAG-Drosha-expressing vectors were co-transfected into HeLa cells with indicated mini-gene vectors, and anti-FLAG-Drosha immunoprecipitates and inputs were analyzed by qRT-PCR for spliced and unspliced melastatin mini-gene transcripts. The efficiency of FLAG-Drosha immunoprecipitation was assessed by Western blotting (p<0.05, Student's t-Test).

(A) A schematic representation of melastatin primary transcript (consisting of 27 exons and 26 introns) producing both miR-211 from intron 6 and spliced melastatin mRNA. Arrow pairs represent qRT-PCR primers used to detect spliced exon-exon junctions. (B) miR-211-containing intron 6 of melastatin is preferentially spliced in primary melanocytes (PM), melanoma patient samples (WM1716 and WM3526), and melanoma cell lines (501mel, UACC62, SKmel2, and 451LU) in a Microprocessor-dependent manner. Relative copy numbers of indicated spliced exon-exon junctions, based on standard curves produced using cloned melastatin cDNA, were determined by qRT-PCR and normalized to Actin before and after Drosha and DGCR8 knockdowns.

The figure of the pathogenic variant in the transient receptor potential melastatin 6 genes