Structure-function analysis of the Yhc1 subunit of yeast U1 snRNP and genetic interactions of Yhc1 with Mud2, Nam8, Mud1, Tgs1, U1 snRNA, SmD3 and Prp28.
Yhc1 and U1C are homologous essential subunits of the yeast and human U1 snRNP, respectively, that are implicated in the establishment and stability of the complex of U1 bound to the pre-mRNA 5' splice site (5'SS). Here, we conducted a mutational analysis of Yhc1, guided by the U1C NMR structure and low-resolution crystal structure of human U1 snRNP. The N-terminal 170-amino acid segment of the 231-amino acid Yhc1 polypeptide sufficed for vegetative growth. Although changing the zinc-binding residue Cys6 to alanine was lethal, alanines at zinc-binding residues Cys9, His24 and His30 were not. Benign alanine substitutions at conserved surface residues elicited mutational synergies with other splicing components. YHC1-R21A was synthetically lethal in the absence of Mud2 and synthetically sick in the absence of Nam8, Mud1 and Tgs1 or in the presence of variant U1 snRNAs. YHC1 alleles K28A, Y12A, T14A, K22A and H15A displayed a progressively narrower range of synergies. R21A and K28A bypassed the essentiality of DEAD-box protein Prp28, suggesting that they affected U1•5'SS complex stability. Yhc1 Arg21 fortifies the U1•5'SS complex via contacts with SmD3 residues Glu37/Asp38, mutations of which synergized with mud2Δ and bypassed prp28Δ. YHC1-(1-170) was synthetically lethal with mutations of all components interrogated, with the exception of Nam8.
Microbiology and Immunology Department, Weill Cornell Medical College, New York, NY 10065, USA and Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.
Beate
B
Schwer
Stewart
S
Shuman
D000409:Alanine / Q000235:genetics; D001665:Binding Sites; D053487:DEAD-box RNA Helicases / Q000235:genetics; D006801:Humans; D008780:Methyltransferases / Q000235:genetics; D009154:Mutation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000737:chemistry; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D000072668:Splicing Factor U2AF; D015032:Zinc / Q000378:metabolism
C089980:MUD2 protein, S cerevisiae; D021122:Protein Subunits; D012342:RNA, Small Nuclear; D017412:Ribonucleoprotein, U1 Small Nuclear; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; C091107:SMD3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000072668:Splicing Factor U2AF; C405483:U1 small nuclear RNA; C075339:U1A protein; C000589307:Yhc1 protein, S cerevisiae; C075023:NAM8 protein, S cerevisiae; D008780:Methyltransferases; C456271:trimethylguanosine synthase; C088939:PRP28 protein, S cerevisiae; D053487:DEAD-box RNA Helicases; D015032:Zinc; D000409:Alanine