Biology subjects

Lipinski, K.

Publications and source records attributed to Lipinski, K..

3 recordsLinked to original sources

bioRxiv2024

Control of 3' splice site selection by the yeast splicing factor Fyv6

Pre-mRNA splicing is catalyzed in two steps: 5' splice site (SS) cleavage and exon ligation. A number of proteins transiently associate with spliceosomes to specifically impact these steps (1st and 2nd step factors). We recently identified Fyv6 (FAM192A in humans) as a 2nd step factor in S. cerevisiae; however, we did not determine how widespread Fyv6s impact is on the transcriptome. To answer this question, we have used RNA-seq to analyze changes in splicing. These results show that loss of Fyv6 results in activation of non-consensus, branch point (BP) proximal 3' SS transcriptome-wide. To identify the molecular basis of these observations, we determined a high-resolution cryo-EM structure of a yeast product complex spliceosome containing Fyv6 at 2.3 [A]. The structure reveals that Fyv6 is the only 2nd step factor that contacts the Prp22 ATPase and that Fyv6 binding is mutually exclusive with that of the 1st step factor Yju2. We then use this structure to dissect Fyv6 functional domains and interpret results of a genetic screen for fyv61 suppressor mutations. The combined transcriptomic, structural, and genetic studies allow us to propose a model in which Yju2/Fyv6 exchange facilitates exon ligation and Fyv6 promotes usage of consensus, BP distal 3' SS.

biochemistry↗

bioRxiv2022

Yeast U6 snRNA made by RNA polymerase II is less stable but functional

U6 small nuclear (sn)RNA is the shortest and most conserved snRNA in the spliceosome and forms a substantial portion of its active site. Unlike the other four spliceosomal snRNAs, which are synthesized by RNA polymerase (RNAP) II, U6 is made by RNAP III. To determine if some aspect of U6 function is incompatible with synthesis by RNAP II, we created a U6 snRNA gene with RNAP II promoter and terminator sequences. This "U6-II" gene is functional as the sole source of U6 snRNA in yeast, but its transcript is much less stable than U6 snRNA made by RNAP III. Addition of the U4 snRNA Sm protein binding site to U6-II increased its stability and led to formation of U6-II*Sm complexes. We conclude that synthesis of U6 snRNA by RNAP III is not required for its function and that U6 snRNPs containing the Sm complex can form in vivo. The ability to synthesize U6 snRNA with RNAP II relaxes sequence restraints imposed by intragenic RNAP III promoter and terminator elements and allows facile control of U6 levels via regulators of RNAP II transcription.

molecular biology↗

bioRxiv2020

Characterization of a new Leishmania major isolate for use in a controlled human infection model

Leishmaniasis is widely regarded as a vaccine-preventable disease, but the costs required to reach pivotal Phase 3 studies and uncertainty about which candidate vaccines should be progressed into human studies significantly limits progress in vaccine development for this neglected tropical disease. Controlled human infection models (CHIM) provide a pathway for accelerating vaccine development and to more fully understand disease pathogenesis and correlates of protection. Here, we describe the isolation, characterization and GMP manufacture of a new clinical isolate of Leishmania major. Two fresh isolates of L. major from Israel were initially compared by genome sequencing, in vivo infectivity and drug sensitivity in mice, and development and transmission competence in sand flies, allowing one (L. major_MRC-02) to be selected for GMP production. This study addresses a major roadblock in the development of vaccines for leishmaniasis, providing a key resource for CHIM studies of sand fly transmitted cutaneous leishmaniasis.

microbiology↗