Regulace sestřihu pre-mRNA v S. cerevisiae: kooperace RNA a proteinů.

Ondřej Gahura, PhD Thesis 2011 Regulation of pre-mRNA splicing in S. cerevisiae: where RNA cooperates with proteins Abstract Removal of introns from protein coding transcripts occurs in two splicing reactions catalyzed by a large nuclear complex, spliceosome. The spliceosome is an extremely intricate and dynamic machine, wherein contributions of small RNA molecules and multiple proteins are coordinated to meet the requirements of absolute precision and high flexibility. For an intimate understanding of pre-mRNA splicing, it is necessary to unravel roles of individual components and to dissect the partial mechanisms. In the first part of this work, we describe the role of the Prp45 splicing factor in Saccharomyces cerevisiae. Mapping of genetic interactions of a conditionally lethal allele prp45(1-169) suggests a relationship of Prp45 to the NTC complex and to the second transesterification. Two-hybrid assay and purification of spliceosomal complexes reveal a contribution of the Prp45 C-terminus in the Prp22 helicase recruitment and/or regulation. Numerous experiments with reporter substrates document the need of Prp45 for the efficient splicing of a specific subset of introns. Our observations suggest that the function of Prp45 in splicing is conserved in evolution. The second part is devoted to the role of...Ondřej Gahura, Dizertační práce 2011 Regulace sestřihu pre-mRNA v S. cerevisiae: kooperace RNA a proteinů Abstrakt Odstraňování intronů z transkriptů probíhá prostřednictvím sestřihu v reakci katalyzované velkým jaderným komplexem - spliceosomem. Sestřih je nesmírně komplikovaný a dynamický proces, v němž koordinované fungování pěti malých molekul RNA a řady proteinů zajišťuje splnění požadavků na extrémní přesnost a flexibilitu. Pro důkladné pochopení sestřihu pre-mRNA je nezbytné rozklíčovat role jednotlivých komponent spliceosomu a porozumět všem dílčím mechanismům. První část práce se zabývá rolí sestřihového faktoru Prp45 v kvasince Saccharomyces cerevisiae. Mapování genetických interakcí alely prp45(1-169) ukazuje na vztah mezi Prp45, NTC komplexem a druhým sestřihovým krokem. Analýza interakcí pomocí dvouhybridního systému a purifikace sestřihových komplexů dokladuje roli C-koncové části Prp45 v regulaci a/nebo vyvazování helikázy Prp22 do spliceosomu. Experimenty s reportérovými substráty prokazují, že Prp45 je vyžadován pro efektivní sestřih určité skupiny intronů. Naše pozorování podporují hypotézu, že role Prp45 v sestřihu je konzervována v evoluci. Druhá část práce je věnována studiu vlivusekundárních struktur intronů na identifikaci 3' sestřihových míst (3' splice site; 3'ss). Ukázali jsme, že...Department of Cell BiologyKatedra buněčné biologieFaculty of SciencePřírodovědecká fakult

ATP synthase from Trypanosoma brucei has an elaborated canonical F1-domain and conventional catalytic sites.

The structures and functions of the components of ATP synthases, especially those subunits involved directly in the catalytic formation of ATP, are widely conserved in metazoans, fungi, eubacteria, and plant chloroplasts. On the basis of a map at 32.5-Å resolution determined in situ in the mitochondria of Trypanosoma brucei by electron cryotomography, it has been proposed that the ATP synthase in this species has a noncanonical structure and different catalytic sites in which the catalytically essential arginine finger is provided not by the α-subunit adjacent to the catalytic nucleotide-binding site as in all species investigated to date, but rather by a protein, p18, found only in the euglenozoa. A crystal structure at 3.2-Å resolution of the catalytic domain of the same enzyme demonstrates that this proposal is incorrect. In many respects, the structure is similar to the structures of F1-ATPases determined previously. The α3β3-spherical portion of the catalytic domain in which the three catalytic sites are found, plus the central stalk, are highly conserved, and the arginine finger is provided conventionally by the α-subunits adjacent to each of the three catalytic sites found in the β-subunits. Thus, the enzyme has a conventional catalytic mechanism. The structure differs from previous described structures by the presence of a p18 subunit, identified only in the euglenozoa, associated with the external surface of each of the three α-subunits, thereby elaborating the F1-domain. Subunit p18 is a pentatricopeptide repeat (PPR) protein with three PPRs and appears to have no function in the catalytic mechanism of the enzyme

Inhibition of F1-ATPase from Trypanosoma brucei by its regulatory protein inhibitor TbIF1.

Hydrolysis of ATP by the mitochondrial F-ATPase is inhibited by a protein called IF1 . In the parasitic flagellate, Trypanosoma brucei, this protein, known as TbIF1 , is expressed exclusively in the procyclic stage, where the F-ATPase is synthesizing ATP. In the bloodstream stage, where TbIF1 is absent, the F-ATPase hydrolyzes ATP made by glycolysis and compensates for the absence of a proton pumping respiratory chain by translocating protons into the intermembrane space, thereby maintaining the essential mitochondrial membrane potential. We have defined regions and amino acid residues of TbIF1 that are required for its inhibitory activity by analyzing the binding of several modified recombinant inhibitors to F1 -ATPase isolated from the procyclic stage of T. brucei. Kinetic measurements revealed that the C-terminal portion of TbIF1 facilitates homodimerization, but it is not required for the inhibitory activity, similar to the bovine and yeast orthologs. However, in contrast to bovine IF1 , the inhibitory capacity of the C-terminally truncated TbIF1 diminishes with decreasing pH, similar to full length TbIF1 . This effect does not involve the dimerization of active dimers to form inactive tetramers. Over a wide pH range, the full length and C-terminally truncated TbIF1 form dimers and monomers, respectively. TbIF1 has no effect on bovine F1 -ATPase, and this difference in the mechanism of regulation of the F-ATPase between the host and the parasite could be exploited in the design of drugs to combat human and animal African trypanosomiases.This work was supported by the Ministry of Education ERC CZ grant LL1205 and Grant Agency of the Czech Republic (18‐17529S) (both to AZ), by European Regional Development Fund (No. CZ.02.1.01/0.0/0.0/16_019/0000759), and by the Medical Research Council of the United Kingdom by Grant 21 522 MC_U1065663150 and by Programme Grant MR/M009858/1 (both to JEW)

Secondary structure is required for 3′ splice site recognition in yeast

Higher order RNA structures can mask splicing signals, loop out exons, or constitute riboswitches all of which contributes to the complexity of splicing regulation. We identified a G to A substitution between branch point (BP) and 3′ splice site (3′ss) of Saccharomyces cerevisiae COF1 intron, which dramatically impaired its splicing. RNA structure prediction and in-line probing showed that this mutation disrupted a stem in the BP-3′ss region. Analyses of various COF1 intron modifications revealed that the secondary structure brought about the reduction of BP to 3′ss distance and masked potential 3′ss. We demonstrated the same structural requisite for the splicing of UBC13 intron. Moreover, RNAfold predicted stable structures for almost all distant BP introns in S. cerevisiae and for selected examples in several other Saccharomycotina species. The employment of intramolecular structure to localize 3′ss for the second splicing step suggests the existence of pre-mRNA structure-based mechanism of 3′ss recognition

Regulation of pre-mRNA splicing in S. cerevisiae: where RNA cooperates with proteins.

Ondřej Gahura, PhD Thesis 2011 Regulation of pre-mRNA splicing in S. cerevisiae: where RNA cooperates with proteins Abstract Removal of introns from protein coding transcripts occurs in two splicing reactions catalyzed by a large nuclear complex, spliceosome. The spliceosome is an extremely intricate and dynamic machine, wherein contributions of small RNA molecules and multiple proteins are coordinated to meet the requirements of absolute precision and high flexibility. For an intimate understanding of pre-mRNA splicing, it is necessary to unravel roles of individual components and to dissect the partial mechanisms. In the first part of this work, we describe the role of the Prp45 splicing factor in Saccharomyces cerevisiae. Mapping of genetic interactions of a conditionally lethal allele prp45(1-169) suggests a relationship of Prp45 to the NTC complex and to the second transesterification. Two-hybrid assay and purification of spliceosomal complexes reveal a contribution of the Prp45 C-terminus in the Prp22 helicase recruitment and/or regulation. Numerous experiments with reporter substrates document the need of Prp45 for the efficient splicing of a specific subset of introns. Our observations suggest that the function of Prp45 in splicing is conserved in evolution. The second part is devoted to the role of..

Synthetic Lethal Interactions of the PRP45 Gene in Saccharomyces cerevisiae

Katedra buněčné biologieDepartment of Cell BiologyPřírodovědecká fakultaFaculty of Scienc

Synthetic Lethal Interactions of the PRP45 Gene in Saccharomyces cerevisiae

Katedra buněčné biologieDepartment of Cell BiologyPřírodovědecká fakultaFaculty of Scienc

TbIF1 functions as a unidirectional inhibitor of <i>T</i>. <i>brucei</i> F_oF₁-ATP synthase.

<p>(A) Total ATPase activity was measured in TbIF1 OE cells that were noninduced (grey) or induced for 2 days with tet (grey cross-hatch). To define the contribution of F_oF₁-ATPase to the total ATPase activity measured, samples were also incubated with either azide (AZ, 1 mM) or oligomycin (OM, 2.5 μg/ml). The total amount of free-phosphate detected in the untreated noninduced sample was set at 100%. (means ± s.d.; n = 3; ** <i>p</i> < 0.001; Student’s <i>t</i> test). (B) The amount of ATP synthesized by oxidative phosphorylation was measured in the digitonin-extracted mitochondria from both noninduced (NON) TbIF1 OE cells and cells induced for 2 days (IND2). The reaction was started by the addition of succinate and ADP. Malonate (mal.) and atractyloside (atract.) were added as specific inhibitors of succinate dehydrogenase and the ATP/ADP carrier, respectively.</p

Recombinant TbIF1 inhibits the F_oF₁-ATPase activity <i>in vitro</i>.

<p>Mitochondria isolated from wildtype PF427 cells were lysed with dodecyl maltoside and the ATPase activity was measured by a Pullman assay. These samples were either treated with azide (AZ, 2 mM), oligomycin (OM, 50 μM) or the indicated rTbIF1 concentrations. (means ± s.d.; n > 3).</p

Neither TbIF1 silencing nor overexpression are harmful to PF <i>T</i>. <i>brucei</i> cells grown <i>in vitro</i>.

<p>(A) TbIF1 RNAi noninduced (NON) and induced (IND) cells were maintained in the exponential growth phase (between 10⁶ and 10⁷ cells/ml) and the cumulative cell number represents the normalization of cell densities by factoring in the daily dilution factor. The figure is representative of three independent RNAi inductions. B) The growth rate of cells either induced (IND) or noninduced (NON) for TbIF1 OE were determined in the same manner as in A. C) The steady-state abundance of TbIF1 in the parental cell line (29–13), TbIF1 RNAi noninduced (NON) and cells induced (IND) with tet for 1, 2, 3 and 4 days was determined by western blot analysis using a specific TbIF1 antiserum. Cytosolic enolase served as a loading control. The numbers depicted underneath the top panel represent the abundance of immunodetected protein as a percentage of the noninduced samples after normalizing to the loading control. D) Ectopic V5-tagged TbIF1 expression was confirmed by western blot analysis using whole cell lysates from PF 29–13, noninduced (NON) TbIF1 OE and cells induced (IND) for 1, 2, 3 and 4 days. The endogenous TbIF1 and the V5-tagged ectopic protein were visualized using a polyclonal TbIF1 antiserum. Comparable loading was confirmed by Bio-Rad TGX stain-free technology. Levels of V5-tagged TbIF1 overexpression as compared to the endogenous TbIF1 are indicated at the top of the immunoblot.</p