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.

Keywords: Luc7; Prp28; U1 snRNP; spliceosome assembly.

FIGURE 1.

Phylogeny-guided mutational analysis of Luc7. The primary structures of the Luc7 polypeptides of Saccharomyces cerevisiae (Sce), Aspergillus fumigatus (Afu), Trichoderma atroviride (Tat), and Homo sapiens (Hsa) are aligned. Positions of side chain identity/similarity are indicated by (•). Gaps in the alignment are denoted by dashes. Forward and reverse arrows indicate the boundaries of S. cerevisiae Luc7 N-terminal and C-terminal truncations, respectively. Black arrows denote biologically active truncations; red arrows indicate lethal truncations. The conserved amino acids of the N-terminal CCCH ZnF1 module are shaded gold; the amino acids of the conserved C-terminal CCHH ZnF2 module are shaded green. Eight other conserved positions subjected to mutagenesis in the present study are shaded magenta.

FIGURE 2.

Deletion and alanine-scanning mutagenesis of yeast Luc7. Truncated LUC7 alleles (top panel), alanine mutants of the cysteines and histidines of the ZnF1 and ZnF2 motifs (middle panels), and additional alanine mutants in the ZnF2 domain (bottom panel) were tested for activity by plasmid shuffle. The viable FOA-resistant luc7Δ strains bearing the indicated LUC7 alleles were spot-tested for growth on YPD agar at the temperatures specified. LUC7 alleles listed at the bottom of each panel failed to complement luc7Δ in the plasmid shuffle assay and were deemed lethal.

FIGURE 3.

Synthetic interactions of Luc7 mutants. Synthetically lethal pairs of alleles are highlighted in red boxes. Other negative pairwise interactions are classified as sick or very sick (yellow boxes) or temperature-sensitive (ts) or cold sensitive (cs) (light green boxes). Gray boxes denote lack of mutational synergy.

FIGURE 4.

Luc7 mutations that bypass Prp28. Yeast prp28Δ luc7Δ cells bearing the indicated LUC7 mutant allele on a CEN LEU2 plasmid were spot-tested for growth on YPD agar at the temperatures specified. Control strains were those that had been cotransformed with plasmids bearing the wild-type PRP28 and LUC7 genes.

FIGURE 5.

Luc7 mutations affect splicing of SUS1 pre-mRNA in vivo. RNA isolated from the indicated LUC7 strains was reverse transcribed with an oligo(dT) primer. cDNAs were PCR-amplified with gene-specific sense and antisense primer pairs derived from the first and last exons of the GLC7 (panel A) and SUS1 (panel B) genes. The exon–intron organization of the pre-mRNAs is shown, with exons depicted as horizontal cylinders. The PCR products were resolved by native agarose gel electrophoresis and visualized by staining with ethidium bromide. The products of PCR amplification of genomic DNA with the same primer pairs are shown in lanes labeled “DNA.” The positions and sizes (bp) of linear duplex DNA markers are indicated on the left. The RT-PCR products of unspliced, partly spliced, and fully spliced transcripts are specified on the right.