Structural basis for recognition of intron branchpoint RNA by yeast Msl5 and selective effects of interfacial mutations on splicing of yeast pre-mRNAs.

Saccharomyces cerevisiae Msl5 orchestrates spliceosome assembly by binding the intron branchpoint sequence 5'-UACUAAC and, with its heterodimer partner protein Mud2, establishing cross intron-bridging interactions with the U1 snRNP at the 5' splice site. Here we define the central Msl5 KH-QUA2 domain as sufficient for branchpoint RNA recognition. The 1.8 Å crystal structure of Msl5-(KH-QUA2) bound to the branchpoint highlights an extensive network of direct and water-mediated protein-RNA and intra-RNA atomic contacts at the interface that illuminate how Msl5 recognizes each nucleobase of the UACUAAC element. The Msl5 structure rationalizes a large body of mutational data and inspires new functional studies herein, which reveal how perturbations of the Msl5·RNA interface impede the splicing of specific yeast pre-mRNAs. We also identify interfacial mutations in Msl5 that bypass the essentiality of Sub2, a DExD-box ATPase implicated in displacing Msl5 from the branchpoint in exchange for the U2 snRNP. These studies establish an atomic resolution framework for understanding splice site selection and early spliceosome dynamics.

The telomere capping complex CST has an unusual stoichiometry, makes multipartite interaction with G-Tails, and unfolds higher-order G-tail structures.

The telomere-ending binding protein complex CST (Cdc13-Stn1-Ten1) mediates critical functions in both telomere protection and replication. We devised a co-expression and affinity purification strategy for isolating large quantities of the complete Candida glabrata CST complex. The complex was found to exhibit a 2∶4∶2 or 2∶6∶2 stoichiometry as judged by the ratio of the subunits and the native size of the complex. Stn1, but not Ten1 alone, can directly and stably interact with Cdc13. In gel mobility shift assays, both Cdc13 and CST manifested high-affinity and sequence-specific binding to the cognate telomeric repeats. Single molecule FRET-based analysis indicates that Cdc13 and CST can bind and unfold higher order G-tail structures. The protein and the complex can also interact with non-telomeric DNA in the absence of high-affinity target sites. Comparison of the DNA-protein complexes formed by Cdc13 and CST suggests that the latter can occupy a longer DNA target site and that Stn1 and Ten1 may contact DNA directly in the full CST-DNA assembly. Both Stn1 and Ten1 can be cross-linked to photo-reactive telomeric DNA. Mutating residues on the putative DNA-binding surface of Candida albicans Stn1 OB fold domain caused a reduction in its crosslinking efficiency in vitro and engendered long and heterogeneous telomeres in vivo, indicating that the DNA-binding activity of Stn1 is required for telomere protection. Our data provide insights on the assembly and mechanisms of CST, and our robust reconstitution system will facilitate future biochemical analysis of this important complex.

Genetic interactions of hypomorphic mutations in the m7G cap-binding pocket of yeast nuclear cap binding complex: an essential role for Cbc2 in meiosis via splicing of MER3 pre-mRNA.

Nuclear cap binding protein complex (CBC) is a heterodimer of a small subunit (Cbc2 in yeast) that binds the m(7)G cap and a large subunit (Sto1 in yeast) that interacts with karyopherins. In order to probe the role of cap recognition in yeast CBC function, we introduced alanine mutations (Y24A, F91A, D120A, D122A, R129A, and R133A) and N-terminal deletions (NΔ21 and NΔ42) in the cap-binding pocket of Cbc2. These lesions had no effect on vegetative growth, but they ameliorated the cold-sensitivity of tgs1Δ cells that lack trimethylguanosine caps (a phenotype attributed to ectopic association of CBC with the m(7)G cap of the normally TMG-capped U1 snRNA), thereby attesting to their impact on cap binding in vivo. Further studies of the Cbc2-Y24A variant revealed synthetic lethality or sickness with null mutations of proteins involved in early steps of spliceosome assembly (Nam8, Mud1, Swt21, Mud2, Ist3, and Brr1) and with otherwise benign mutations of Msl5, the essential branchpoint binding protein. Whereas the effects of weakening CBC-cap interactions are buffered by other actors in the splicing pathway during mitotic growth, the NΔ42 allele causes a severe impediment to yeast sporulation and meiosis. RNA analysis revealed a selective defect in the splicing of MER3 and SAE3 transcripts in cbc2-NΔ42 diploids during attempted sporulation. An intronless MER3 cDNA fully restored sporulation and spore viability in the cbc2-NΔ42 strain, signifying that MER3 splicing is a limiting transaction. These studies reveal a new level of splicing control during meiosis that is governed by nuclear CBC.

The Candida albicans Ku70 modulates telomere length and structure by regulating both telomerase and recombination.

The heterodimeric Ku complex has been shown to participate in DNA repair and telomere regulation in a variety of organisms. Here we report a detailed characterization of the function of Ku70 in the diploid fungal pathogen Candida albicans. Both ku70 heterozygous and homozygous deletion mutants have a wild-type colony and cellular morphology, and are not sensitive to MMS or UV light. Interestingly, we observed complex effects of KU70 gene dosage on telomere lengths, with the KU70/ku70 heterozygotes exhibiting slightly shorter telomeres, and the ku70 null strain exhibiting long and heterogeneous telomeres. Analysis of combination mutants suggests that the telomere elongation in the ku70 null mutant is due mostly to unregulated telomerase action. In addition, elevated levels of extrachromosomal telomeric circles were detected in the null mutant, consistent with activation of aberrant telomeric recombination. Altogether, our observations point to multiple mechanisms of the Ku complex in telomerase regulation and telomere protection in C. albicans, and reveal interesting similarities and differences in the mechanisms of the Ku complex in disparate systems.

Telomerase regulatory subunit Est3 in two Candida species physically interacts with the TEN domain of TERT and telomeric DNA.

The yeast telomerase regulatory protein Est3 is required for telomere maintenance in vivo, and shares intriguing structural and functional similarities with the mammalian telomeric protein TPP1. Here we report our physical and functional characterizations of Est3 homologues from Candida parapsilosis and Lodderomyces elongisporus, which bear unique N- and C-terminal tails in addition to a conserved central OB fold. We show that these Est3 homologues form stable complexes with the TEN domain of telomerase reverse transcriptase. Efficient complex formation requires both the N- and C-terminal tails, as well as conserved OB fold residues of Est3. Other Est3 homologues devoid of the tails failed to interact strongly with the cognate TEN domains. Remarkably, the C. parapsilosis Est3 alone exhibits no appreciable DNA-binding activity, but can be crosslinked to telomeric DNA in the presence of the TEN domain. A conserved basic residue on the putative DNA-binding surface of CpEst3 is required for efficient crosslinking. Mutating the equivalent residue in Candida albicans Est3 caused telomere attrition. We propose that interaction with the TEN domain unmasks a functionally important nucleic acid-binding activity in Est3. Our findings provide insights on the mechanisms and evolution of a widely conserved and functionally critical telomeric/telomerase component.