FIGURE 1.

Gemin5 binds to the mature SMN mRNA in vivo and in vitro. RT-PCR of modified PAR-CLIP to detect SMN mRNA bound to Gemin5 in both (A) HeLa and (B) MN1 cell extract. Control is an isotype-matched IgG antibody. C, diagram of RNA transcripts used in D and E. The SMN pre-mRNA construct is 192 nucleotides in length and 118 nucleotides for the mature mRNA exclusive of the 5 nucleotide pA tail. The regions deleted in subsequent experiments are indicated by H2, H1, as well as the U-rich region (white text). Further 10 or 20 nucleotide deletions were made to the 3′-end as indicated. D, Western blot of mutant and wild type SMN mRNA fragments in streptavidin pulldown assays. Control RNA used is a 60-nucleotide nonspecific RNA transcribed from empty pSP72. E, (left) Western blot of mutant and wild type SMN probing for proteins of the SMN complex in a streptavidin pulldown assay. Right, Western blot of SMN immunoprecipitation showing expected ratios of complex proteins. Control is the same as in D. For all, input represents extract from cell lines. IP, immunoprecipitation; Δ10 and Δ20, deletion of 10 or 20 nucleotides; wt, wild type; H2, helical region 2; H1, helical region 1.