Figure 9

U1 snRNP binds mature mRNAs. (a) Putative base pairs between the 5′ end of U1 snRNA and the CG3776 mRNA coding region (upper panel). Within the putative region of base pairing, three translationally silent point mutations were introduced (bold blue letters) to disrupt the helix (lower panel). (b) Cartoon of the S2 cell transfection construct, showing the CG3776 expression unit. CG3776endo and CG3776tag indicate locations of primers for qRT-PCR. CG3776endo amplifies both endogenous and transfected CG3776 mRNAs, whereas CG3776tag amplifies transfected CG3776 mRNA only. The black star indicates the location of the putative U1 binding site. (c) pAW vector, pAW-CG3776wt and pAW-CG3776mut were transfected into S2 cells, and CG3776wt and CG3776mut expression was measured using qRT-PCR with the CG3776endo primer pair. GAPDH was used as normalization standard. (d) After pAW-CG3776wt and pAW-CG3776mut were transfected, anti-Sm (Y12) IPs were performed using S2 cell lysate. GAPDH was used as normalization standard. (e) Proposed model of snRNP-mRNA interactions. Distinct snRNPs (U1 and potentially others) associate with mature mRNAs via base pairing and/or protein-mediated interaction. Such interactions could serve as a platform to recruit RNA processing factors that act on multiple levels of RNA metabolism. t-Test for significance between IP and control (Ctrl): *P < 0.05, **P < 0.01, ***P < 0.001. Mut, mutant; wt, wild-type.