Abstract
Unlike most other eukaryotes, Leishmania and other trypanosomatid protozoa have largely eschewed transcriptional control of gene expression, relying instead on post-transcriptional regulation of mRNAs derived from polycistronic transcription units (PTUs). In these parasites, a novel modified nucleotide base (β-D-glucopyranosyloxymethyluracil) known as J plays a critical role in ensuring that transcription termination occurs only at the end of each PTU, rather than at the polyadenylation site of individual genes. To further understand the biology of J-associated processes, we used tandem affinity purification (TAP-tagging) and mass spectrometry to reveal proteins that interact with the glucosyltransferase performing the final step in J synthesis. These studies identified four proteins reminiscent of subunits in the PTW/PP1 complex that controls transcription termination in higher eukaryotes. Moreover, bioinformatics analyses identified the DNA-binding subunit of Leishmania PTW/PP1 as a novel J-binding protein, which we dubbed JBP3. Down-regulation of JBP3 expression levels in Leishmania results in a substantial increase in transcriptional read-through at the 3’ end of most PTUs. Additional TAP-tagging experiments showed that JBP3 also associates with two other protein complexes. One consists of subunits with domains suggestive of a role in chromatin modification/remodeling, while the other contains subunits with similarity to those found in the PAF1C complex involved in regulation of transcription in other eukaryotes. Thus, while trypanosomatids utilize protein complexes similar to that used to control transcription termination in other eukaryotes, JBP3 appears to function as a hub linking these modules to base J, thereby enabling the parasites’ unique reliance on polycistronic transcription and post-transcriptional regulation of gene expression.
Competing Interest Statement
The authors have declared no competing interest.