Structure–function analysis and genetic interactions of the Luc7 subunit of the Saccharomyces cerevisiae U1 snRNP

Abstract

Luc7 is an essential 261-amino acid protein subunit of the Saccharomyces cerevisiae U1 snRNP. To establish structure-function relations for yeast Luc7, we conducted an in vivo mutational analysis entailing N-and C-terminal truncations and alanine scanning of phylogenetically conserved amino acids, including two putative zinc finger motifs, ZnF1 and ZnF2, and charged amino acids within the ZnF2 module. We identify Luc7-(31-246) as a minimal functional protein and demonstrate that whereas mutations of the CCHH ZnF2 motif are lethal, mutations of the ZnF1 CCCH motif and the charged residues of the ZnF2 modules are not. Though dispensable for vegetative growth in an otherwise wild-type background, the N-terminal 18-amino acid segment of Luc7 plays an important role in U1 snRNP function, evinced by our findings that its deletion (i) impaired the splicing of SUS1 pre-mRNA; (ii) was synthetically lethal absent other U1 snRNP constituents (Mud1, Nam8, the TMG cap, the C terminus of Snp1), absent the Mud2 subunit of the Msl5•Mud2 branchpoint binding complex, and when the m 7 G cap-binding site of Cbc2 was debilitated; and (iii) bypassed the need for the essential DEAD-box ATPase Prp28. Similar phenotypes were noted for ZnF1 mutations C45A, C53A, and C68A and ZnF2 domain mutations D214A, R215A, R216A, and D219A. These findings highlight the contributions of the Luc7 N-terminal peptide, the ZnF1 motif, and the ZnF2 module in stabilizing the interactions of the U1 snRNP with the pre-mRNA 5 ′ splice site and promoting the splicing of a yeast pre-mRNA, SUS1, that has a nonconsensus 5 ′ splice site.

Structures of the fully assembled Saccharomyces cerevisiae spliceosome before activation

“…3B). These general structural features corroborate the reported biochemical functions of Yhc1 and Luc7 (24)(25)(26). Yhc1 (U1-C in humans) is an essential subunit of U1 snRNP and plays an important role in stabilizing the 5′SS/U1 duplex (24,(27)(28)(29).…”

“…A C-terminal fragment (residues 198 to 230) of Luc7 also forms a C2H2-type zinc finger ( fig. S9, B and C), which is known to promote the splicing of pre-mRNA with a weak 5′SS (26,30,31). In our structure, three charged residues from this zinc finger-Asp 212 , Arg 216 , and Lys 224 -directly contact the 5′SS/U1 duplex through H-bonds (Fig.…”

Defining essential elements and genetic interactions of the yeast Lsm2–8 ring and demonstration that essentiality of Lsm2–8 is bypassed via overexpression of U6 snRNA or the U6 snRNP subunit Prp24

“…SUS1 splicing is adversely affected by a mutation in the m 7 G cap binding pocket of the nuclear cap-binding protein Cbc2 (Qiu et al 2012), by mutations in the branchpoint binding protein Msl5 that perturb the Msl5•branchpoint RNA interface (Jacewicz et al 2015), and by mutations in the U1 snRNP subunit Luc7 (Agarwal et al 2016). SUS1 is one of a handful of yeast genes that contain two introns, and it is the splicing of the first intron (which has a nonconsensus 5 ′ splice site GUAUGA and a nonconsensus branchpoint sequence UACUGAC) that is selectively impaired in the aforementioned strain backgrounds (Hossain et al 2009;Qiu et al 2012).…”

Structure–function analysis and genetic interactions of the SmG, SmE, and SmF subunits of the yeast Sm protein ring

“…Genetic analyses in yeast have revealed an extensive network of buffered functions during spliceosome assembly, defined by the numerous instances in which null alleles of vegetatively inessential splicing factors, or benign mutations in essential players, elicit synthetic lethal and sick phenotypes when combined with other benign mutations in the splicing machinery (Liao et al 1991;Abovich et al 1994;Colot et al 1996;Gottschalk et al 1998;Hausmann et al 2008;Wilmes et al 2008;Costanzo et al 2010;Chang et al 2012;Qiu et al 2012;Schwer et al 2013;Shuman 2014, 2015;Jacewicz et al 2015;Agarwal et al 2016). The present collection of biologically active mutants targeted to conserved SmG, SmE, and SmF amino acids at their RNA binding sites or subunit interfaces enables us to survey genetic interactions of these three Sm ring subunits.…”