Membrane proteins are essential for many cell processes yet are more difficult to investigate than soluble proteins. Charged residues often contribute significantly to membrane protein function. Model peptides such as GWALP23 (acetyl‐GGALW5LAL8LALALAL16ALW19LAGA‐amide) can be used to characterize the influence of specific residues on transmembrane protein domains. We have substituted R8 and R16 in GWALP23 in place of L8 and L16, equidistant from the peptide center, and incorporated specific 2H‐labeled alanine residues within the central sequence for detection by solid‐state 2H NMR spectroscopy. The resulting pattern of [2H]Ala quadrupolar splitting (Δνq) magnitudes indicates the core helix for R8,16GWALP23 is significantly tilted to give a similar transmembrane orientation in thinner bilayers with either saturated C12:0 or C14:0 acyl chains (1,2‐dilauroyl‐sn‐glycero‐3‐phosphocholine (DLPC) or 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC)) or unsaturated C16:1 Δ9 cis acyl chains. In bilayers of 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC; C18:1 Δ9 cis) multiple orientations are indicated, whereas in longer, unsaturated 1,2‐dieicosenoyl‐sn‐glycero‐3‐phosphocholine (DEiPC; C20:1 Δ11 cis) bilayers, the R8,16GWALP23 helix adopts primarily a surface orientation. The inclusion of 10–20 mol % cholesterol in DOPC bilayers drives more of the R8,16GWALP23 helix population to the membrane surface, thereby allowing both charged arginines access to the interfacial lipid head groups. The results suggest that hydrophobic thickness and cholesterol content are more important than lipid saturation for the arginine peptide dynamics and helix orientation in lipid membranes.

Precursor mRNA (pre-mRNA) splicing is an essential process for gene expression in eukaryotes catalyzed by the spliceosome in two transesterification steps. The spliceosome is a large, highly dynamic complex composed of 5 small nuclear RNAs and dozens of proteins, some of which are needed throughout the splicing reaction while others only act during specific stages. The human protein FAM192A was recently proposed to be a splicing factor that functions during the second transesterification step, exon ligation, based on analysis of cryo-electron microscopy (cryo-EM) density. It was also proposed that Fyv6 might be the functional S. cerevisiae homolog of FAM192A; however, no biochemical or genetic data has been reported to support this hypothesis. Herein, we show that Fyv6 is a splicing factor and acts during exon ligation. Deletion of FYV6 results in genetic interactions with the essential splicing factors Prp8, Prp16, and Prp22; decreases splicing in vivo of reporter gene harboring intron substitutions that limit the rate of exon ligation; and changes 3' splice site (SS) selection. Together, these data suggest that Fyv6 is a component of the spliceosome and the potential functional and structural homolog of human FAM192A.

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U6 small nuclear (sn)RNA is the shortest and most conserved snRNA in the spliceosome and forms a substantial portion of its active site. Unlike the other four spliceosomal snRNAs, which are synthesized by RNA polymerase (RNAP) II, U6 is made by RNAP III. To determine if some aspect of U6 function is incompatible with synthesis by RNAP II, we created a U6 snRNA gene with RNAP II promoter and terminator sequences. This “U6-II” gene is functional as the sole source of U6 snRNA in yeast, but its transcript is much less stable than U6 snRNA made by RNAP III. Addition of the U4 snRNA Sm protein binding site to U6-II increased its stability and led to formation of U6-II•Sm complexes. We conclude that synthesis of U6 snRNA by RNAP III is not required for its function and that U6 snRNPs containing the Sm complex can form in vivo. The ability to synthesize U6 snRNA with RNAP II relaxes sequence restraints imposed by intragenic RNAP III promoter and terminator elements and allows facile control of U6 levels via regulators of RNAP II transcription.

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