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

Abstract

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.

Inhibitory Receptor Expression on CD8+ T Cells Is Linked to Functional Responses against Trypanosoma cruzi Antigens in Chronic Chagasic Patients

“…After reaching semiconfluence, the VERO cells were incubated for 10 h with T. cruzi I trypomastigotes (MHOM/CO/01/DA human isolate) (34), and the parasites were recovered at 96 h postinfection (35,36). A trypomastigote lysate was obtained as previously described (37). In brief, the parasite culture was washed twice with cold 13 PBS (Eurobio), pH 7.0, and suspended at 1 3 10 6 parasites/ml in lysis buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Nonidet P-40, and 1.4% Triton X-100) containing 2% protease inhibitor mixture (Sigma-Aldrich, St. Louis, MO).…”

RNA Polymerase Transcription Machinery in Trypanosomes

“…It is important to discover which of the RPB6 and RPB10 paralogs reside in the trypanosome RNA pol III enzyme. Additionally, it will be interesting to see if the division of RBP5, RPB6, and RPB10 paralogs is the same in all trypanosomes or in T. brucei alone, where pol II shares the functions of pre-mRNA synthesis with RNA pol I (37).…”

Trypanosome cdc2-Related Kinase 9 Controls Spliced Leader RNA cap4 Methylation and Phosphorylation of RNA Polymerase II Subunit RPB1

“…SLRNA transcription is monocistronic, initiates at a distinct initiation site, and requires the assembly of a conventional, albeit extremely divergent, preinitiation complex (16)(17)(18). The T. brucei genome encodes a full set of RNA Pol II subunits, termed RPB1 to RPB12 (19), and biochemical characterization of the enzyme complex identified all subunits except RPB10 (20)(21)(22). In T. brucei, there are two slightly different RPB1 genes (relevant T. brucei gene accession numbers are listed in Table S1 in the supplemental material) that appear to be functionally redundant, since deletion of three RPB1 alleles did not impair trypanosome proliferation in culture (23).…”