Roles of the Pumilio domain protein PUF3 inTrypanosoma bruceigrowth and differentiation

Abstract

Trypanosomes strongly rely on post-transcriptional mechanisms to control gene expression. Several Opisthokont Pumilio domain proteins are known to suppress expression when bound to mRNAs. The Trypanosoma brucei Pumilio domain protein PUF3 is a cytosolic mRNA-binding protein that suppresses expression when tethered to a reporter mRNA. RNA-binding studies showed that PUF3 preferentially binds to mRNAs with a classical Pumilio-domain recognition motif, UGUA[U/C]AUU. RNA-interference-mediated reduction of PUF3 in bloodstream forms caused a minor growth defect, but the transcriptome was not affected. Depletion of PUF3 also slightly delayed differentiation to the procyclic form. However, both PUF3 genes could be deleted in cultured bloodstream- and procyclic-form trypanosomes. Procyclic forms without PUF3 also grew somewhat slower than wild-type, but ectopic expression of C-terminally tagged PUF3 impaired their viability. PUF3 was not required for RBP10-induced differentiation of procyclic forms to bloodstream forms. Mass spectrometry revealed no PUF3 binding partners that might explain its suppressive activity. We conclude that PUF3 may have a role in fine-tuning gene expression. Since PUF3 is conserved in all Kinetoplastids, including those that do not infect vertebrates, we suggest that it might confer advantages within the invertebrate host.

PUF3 RNA binding protein of Trypanosoma cruzi regulates mitochondrial morphology and function

“…S4C). This may suggest that the correct level of PUF3 is required for optimal growth and that an excess might be toxic, as was shown in the related parasite, T. brucei [ 24 ] and in S. cerevisiae [ 21 ].…”

“…Recently, the PUF3 protein was evaluated in this parasite; the study concluded that PUF3 may have a role in fine-tuning gene expression since RNA-interference PUF3 bloodstream forms had only minor growth defects, and the transcriptome was unaffected. Furthermore, procyclic forms without PUF3 grew slower, but ectopic expression of PUF3 impaired their viability, suggesting a toxic effect of the protein [ 24 ].…”

In vitro culture of freshly isolated Trypanosoma brucei brucei bloodstream forms results in gene copy-number changes

“…Starting with the same initial total RNA samples, we made mRNA by oligo d(T) selection, or by depleting rRNA using RNase H and complementary oligonucleotides. We had previously demonstrated, by comparing with total RNA, that the latter procedure has no effect on the transcriptome [25]. Analysis of the transcriptomes clearly showed that oligo d(T) selection causes loss of longer RNAs (S1C Fig).…”

A bioinformatic approach for the prediction and functional classification ofToxoplasma gondiilong non-coding RNAs

“…Moreover, we observed Pumilio binding motifs at the predicted TglncRNA sequence that let us infer that the sequence would be functional 45 . Although Pumilio proteins were characterized in T. gondii and other parasites as regulators of translation by mRNA binding during differentiation processes [61][62][63] , no role at genome stability was proposed. In fact, a motif binding search for PUM proteins at T. gondii 5'-UTR and 3'-UTR transcripts reveals mRNA targets like those that codify for DNA repair proteins.…”