Characterization of RNA polymerase II subunits of Trypanosoma brucei

Devaux, Sara; Lecordier, Laurence; Uzureau, Pierrick; Walgraffe, David; Dierick, Jean‐François; Poelvoorde, Philippe; Pays, Étienne; Vanhamme, Luc

doi:10.1016/j.molbiopara.2006.02.020

Cited by 30 publications

(57 citation statements)

References 56 publications

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“…In the experiments presented here, we have identified nine subunits (RPB1, RPB2, RPB3, RPB5, RPB6, RPB7, RPB8, RPB10 and RPB11) that co-purify with LmRPB2-TAP and/or LmRPB6-TAP (Table 1). This confirms two recent reports that also identified these subunits (except RPB10) in the T. brucei RNAP II complex isolated by TAPtagging TbRPB9 (Devaux et al, 2006) or TbRPB4 (Das et al, 2006). The original BLAST analyses of the Tritryp genomes revealed only 11 of the 17 RNAP III subunits (Ivens et al, 2005), with most of the RNAP III-specific subunits missing.…”

Section: Discussionsupporting

confidence: 87%

“…Immunoprecipitation using protein C-tagged RPA1 (Schimanski et al, 2003;Nguyen et al, 2006) and protein A-tagged RPA12 (Walgraffe et al, 2005) confirmed the presence of RPA1, RPA2, RPA12, RPAC1, RPAC2, RPB5z (also called 1RPB5), RPB6z (1RPB6), RPB10z (1RPB10) and RPB8 in the T. brucei RNAP I. Similar experiments using tandem affinity purification (TAP)-tagged RPB9 (Devaux et al, 2006) and RPB4 (Das et al, 2006) have recently identified the T. brucei RPB1, RPB2, RPB3, RPB4, RPB5, RPB6, RPB7, RPB8, RPB9 and RPB11 subunits and confirmed that the RNAP I and RNAP II complexes contain different subunits of RPB5.…”

Section: Introductionmentioning

confidence: 66%

“…However, the TriTryp genomes contain two distinct paralogues of RPB5, RPB6 and RPB10 (Ivens et al, 2005;Kelly et al, 2005;Nguyen et al, 2006), and previous reports in T. brucei indicate that at least the RPB5 and RPB6 isoforms are segregated between RNAP I and RNAP II (Walgraffe et al, 2005;Nguyen et al, 2006;Devaux et al, 2006;Das et al, 2006). Our TAP-tag purifications with LmRPB6 as bait confirm these results and indicate that the RPB6 and RPB5 isoforms are found within both the RNAP II and RNAP III complexes, while the RPB6z and RPB5z isoforms are found only in RNAP I.…”

Section: Discussionmentioning

confidence: 93%

“…In T. brucei this procedure (with modifications in some cases) has been successfully used to analyze the exosome complex (Estevez et al, 2001), to identify members of the RNA editing complex (Panigrahi et al, 2003), to study the composition of RNAP I (Schimanski et al, 2003;Walgraffe et al, 2005;Nguyen et al, 2006) and RNAP II (Devaux et al, 2006;Das et al, 2006) complexes, to determine the composition of U1 small nuclear RNP (Palfi et al, 2005), and to identify proteins involved in synthesis of the spliced-leader RNA (Das et al, 2005;Schimanski et al, 2005). Similarly, in Leishmania tarentolae this protocol has been employed to analyze the editosome complex (Aphasizhev et al, 2003b).…”

Section: Discussionmentioning

confidence: 99%

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Characterization of the RNA polymerase II and III complexes in Leishmania major

Martínez‐Calvillo

Saxena

Green

et al. 2007

International Journal for Parasitology

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Transcription of protein-coding genes in Leishmania major and other trypanosomatids differs from that in most eukaryotes and bioinformatic analyses have failed to identify several components of the RNA polymerase (RNAP) complexes. To increase our knowledge about this basic cellular process, we used tandem affinity purification (TAP) to identify subunits of RNAP II and III. Mass spectrometric analysis of the complexes co-purified with TAP-tagged LmRPB2 (encoded by LmjF31.0160) identified seven RNAP II subunits: RPB1, RPB2, RPB3, RPB5, RPB7, RPB10 and RPB11. With the exception of RPB10 and RPB11, and the addition of RPB8, these were also identified using TAP-tagged constructs of one (encoded by LmjF34.0890) of the two LmRPB6 orthologues. The latter experiments also identified the RNAP III subunits RPC1 (C160), RPC2 (C128), RPC3 (C82), RPC4 (C53), RPC5 (C37), RPC6 (C34), RPC9 (C17), RPAC1 (AC40) and RPAC2 (AC19). Significantly, the complexes precipitated by TAP-tagged LmRPB6 did not contain any RNAP I-specific subunits, suggesting that, unlike in other eukaryotes, LmRPB6 is not shared by all three polymerases but is restricted to RNAP II and III, while the LmRPB6z (encoded by LmjF25.0140) isoform is limited to RNAP I. Similarly, we identified peptides from only one (encoded by LmjF18.0780) of the two RPB5 orthologues and one (LmjF13.1120) of the two RPB10 orthologues, suggesting that LmRPB5z (LmjF18.0790) and LmRPB10z (LmjF13.1120) are also restricted to RNAP I. In addition to these RNAP subunits, we also identified a number of other proteins that co-purified with the RNAP II and III complexes, including a potential transcription factor, several histones, an ATPase involved in chromosome segregation, an endonuclease, four helicases, RNA splicing factor PTSR-1, at least two RNA binding proteins and several proteins of unknown function.

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Section: Discussionsupporting

confidence: 87%

Section: Introductionmentioning

confidence: 66%

Section: Discussionmentioning

confidence: 93%

Section: Discussionmentioning

confidence: 99%

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Characterization of the RNA polymerase II and III complexes in Leishmania major

Martínez‐Calvillo

Saxena

Green

et al. 2007

International Journal for Parasitology

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“…They were grown at 37°C with 5% CO 2 in HMI-9 medium (50) supplemented with 10% (vol/vol) FBS (Sigma) and 10% (vol/vol) SERUM PLUS (SAFC Biosciences). Electroporation and selection procedures were conducted as described previously (51).…”

Section: Methodsmentioning

confidence: 99%

Transcription is initiated on silent variant surface glycoprotein expression sites despite monoallelic expression in Trypanosoma brucei

Kassem

Pays

Vanhamme

2014

Proc. Natl. Acad. Sci. U.S.A.

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Significance African trypanosomes are a major plague in sub-Saharan Africa. They cause sleeping sickness in humans and nagana in cattle, preventing extensive cattle raising. They escape the immune system of their host through frequent changes in their major surface antigen, the variant surface glycoprotein (VSG). The control of VSG expression is poorly understood, and the level at which it takes place has been a matter of debate for the last 25 y. A better understanding of the mechanism by which it works would facilitate the identification of the proteins involved and would provide putative targets for drug design. We report data indicating an unusual control level, namely after transcription initiation, suggesting transcription elongation or RNA processing as a control step.

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Absence of the Rpb9 subunit of RNA polymerase II reduces the chronological life span in fission yeast

Bhardwaj

Sharma

2022

J Basic Microbiol

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Fission yeast RNA polymerase II consists of 12 subunits, Rpb1-Rpb12. Among these subunits, Rpb9 is the only subunit whose absence does not cause lethality under optimum growth conditions in fission yeast. However, an rpb9 null fission yeast mutant exhibits a slow-growth phenotype under optimum growth conditions and a defect in survival under environmental and genotoxic stress conditions. To further

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Characterization of RNA polymerase II subunits of Trypanosoma brucei

Cited by 30 publications

References 56 publications

Characterization of the RNA polymerase II and III complexes in Leishmania major

Characterization of the RNA polymerase II and III complexes in Leishmania major

Transcription is initiated on silent variant surface glycoprotein expression sites despite monoallelic expression in Trypanosoma brucei

Absence of the Rpb9 subunit of RNA polymerase II reduces the chronological life span in fission yeast

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