title: 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.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA.
authors.forename: Radhika
authors.initials: R
authors.lastname: Agarwal
authors.affiliation: Microbiology and Immunology Department, Weill Cornell Medical College, New York, New York 10065, USA.
authors.forename: Beate
authors.initials: B
authors.lastname: Schwer
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA.
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017381:Spliceosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072668:Splicing Factor U2AF / Q000235:genetics / Q000378:metabolism; D016335:Zinc Fingers
chemical_list: C534792:Luc7 protein, S cerevisiae; C089980:MUD2 protein, S cerevisiae; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; D000072668:Splicing Factor U2AF; C481300:Sus1 protein, S cerevisiae
keywords: Luc7; Prp28; U1 snRNP; spliceosome assembly