Stress Induces Changes in the Phosphorylation of Trypanosoma cruzi RNA Polymerase II, Affecting Its Association with Chromatin and RNA Processing

Abstract

The phosphorylation of the carboxy-terminal heptapeptide repeats of the largest subunit of RNA polymerase II (Pol II) controls several transcription-related events in eukaryotes. Trypanosomatids lack these typical repeats and display an unusual transcription control. RNA Pol II associates with the transcription site of the spliced leader (SL) RNA, which is used in the trans-splicing of all mRNAs transcribed on long polycistronic units. We found that Trypanosoma cruzi RNA Pol II associated with chromatin is highly phosphorylated. When transcription is inhibited by actinomycin D, the enzyme runs off from SL genes, remaining hyperphosphorylated and associated with polycistronic transcription units. Upon heat shock, the enzyme is dephosphorylated and remains associated with the chromatin. Transcription is partially inhibited with the accumulation of housekeeping precursor mRNAs, except for heat shock genes. DNA damage caused dephosphorylation and transcription arrest, with RNA Pol II dissociating from chromatin although staying at the SL. In the presence of calyculin A, the hyperphosphorylated form detached from chromatin, including the SL loci. These results indicate that in trypanosomes, the unusual RNA Pol II is phosphorylated during the transcription of SL and polycistronic operons. Different types of stresses modify its phosphorylation state, affecting pre-RNA processing.

Identification of a novel base J binding protein complex involved in RNA polymerase II transcription termination in trypanosomes

“…However, the CTD was shown to be essential for Pol II transcription in T. brucei [102,103] and 17 phosphorylated sites have been identified within the CTD [100]. Studies also suggest that CTD phosphorylation is required for Pol II association with trypanosome chromatin [104]. It is necessary to determine the role of the PJW/PP1 complex in the phosphorylation status of Spt5 and Pol II in trypanosomatids and whether phosphorylation of a non-canonical Pol II CTD is involved in transcriptional regulation in divergent eukaryotes thought to lack significant regulation.…”

Regulation of gene expression in trypanosomatids: living with polycistronic transcription

“…After a severe heat shock (41°C), trypanosomes shut down transcription initiation [293]. In T. cruzi , this is accompanied (or perhaps caused) by RNA polymerase II large subunit dephosphorylation [294]. Translation of most T. brucei mRNAs is also suppressed, by an unknown mechanism, with either transfer to stress granules or degradation [295].…”

Rapid block of pre‐mRNA splicing by chemical inhibition of analog‐sensitive CRK9 in Trypanosoma brucei

“…In addition, upon CRK9 AS1 ‐PTP inhibition, only RNA pol II‐synthesized pre‐mRNA decreased after the 3 hr time point (Figure 5a), suggesting a synthesis defect. This notion was backed up by a study in T. cruzi in which stress‐induced CTD dephosphorylation led to release of RNA pol II from chromatin (Rocha, Moretti, & Schenkman, 2014). Surprisingly though, in permeabilized trypanosomes in which CRK9 AS1 ‐PTP had been inhibited for 24 hr, RNA pol II‐mediated ATUB and PAP1 transcription was significantly upregulated (Figure 7).…”