Characterization of subunits of the RNA polymerase I complex in Trypanosoma brucei

Walgraffe, David; Devaux, Sara; Lecordier, Laurence; Dierick, Jean‐François; Dieu, Marc; Abbeele, Jan Van Den; Pays, Étienne; Vanhamme, Luc

doi:10.1016/j.molbiopara.2004.11.014

Cited by 32 publications

(45 citation statements)

References 37 publications

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“…We believe the experiments described in this study represent the first comprehensive experimental characterization of the RNAP III complex from the trypanosomatids and confirm the composition of the RNAP II complex previously characterized in T. brucei (Devaux et al, 2006;Das et al, 2006). These results, together with similar studies for T. brucei RNAP I (Schimanski et al, 2003;Walgraffe et al, 2005;Nguyen et al, 2006) significantly expand our knowledge of the unique transcriptional machinery in these primitive parasitic protozoa.…”

Section: Discussionsupporting

confidence: 83%

“…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: 92%

“…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%

“…The paralogous copies are widely divergent, suggesting that the subunits they encode may not be shared by the different RNAP complexes, as they are in other organisms. 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: 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: 83%

Section: Discussionmentioning

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

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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“…The efficiency of the TAP method in purification of protein complexes and identification of interactions was also tested in other organisms, including Dictyostelium (Koch et al, 2006;Meima et al, 2007), Trypanosoma bruce (Mani et al, 2011;Nguyen et al, 2007;Palfi et al, 2005;Walgraffe et al, 2005) and Plasmodium falciparum (Takebe et al, 2007).…”

Section: Tap In Other Organismsmentioning

confidence: 99%

Modification, Development, Application and Prospects of Tandem Affinity Purification Method

Xu¹,

Li²,

Zhang³

et al. 2012

Protein Interactions

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The RNA polymerase I subunit Rpa12p interacts with the stress‐responsive transcription factor Msn4p to regulate lipid metabolism in budding yeast

et al. 2016

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In Saccharomyces cerevisiae, RPA12 encodes the small subunit of RNA polymerase I. Here, we demonstrate that Rpa12p interacts with the transcription factor Msn4p and prevents its binding to the promoter of AYR1 encoding Ayr1p (1-acyldihydroxyacetone phosphate reductase), a key enzyme involved in triacylglycerol biosynthesis and mobilization of nonpolar lipids. Deletion of RPA12 leads to triacylglycerol accumulation due to the binding of Msn4p to the promoter of AYR1 and activation of its transcription. The double deletion rpa12Δ::ayr1Δ caused a reduction in triacylglycerol levels. Our findings reveal that Rpa12p functions as a negative regulator of lipid metabolism by modulating nonpolar lipid biosynthesis through its interaction with Msn4p.

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Characterization of subunits of the RNA polymerase I complex in Trypanosoma brucei

Cited by 32 publications

References 37 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

Modification, Development, Application and Prospects of Tandem Affinity Purification Method

The RNA polymerase I subunit Rpa12p interacts with the stress‐responsive transcription factor Msn4p to regulate lipid metabolism in budding yeast

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