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.

Members of the Puf family of RNA-binding proteins from Drosophila, Caenorhabditis elegans, and Dictyostelium are known to function as translational repressors. To identify mammalian proteins that might regulate posttranscriptional gene expression, we have characterized a novel murine Puf protein, PUM2. Pum2 transcripts were expressed in all murine tissues examined, suggesting the gene influences processes common to many cell types. Like all Puf family members, PUM2 contains a C-terminal RNA-binding domain related to the Drosophila Pumilio homology domain (PUM-HD). Two features found in the amino-terminus of PUM2, regions rich in serine and glutamine/alanine-rich regions, were also identified in most Puf family members. RNA sequences capable of binding with high affinity (6.5 nM) to a 48-kDa recombinant protein containing the PUM2 PUM-HD were isolated by using an iterative amplification–selection protocol (SELEX). The consensus sequence [UGUANAUARNNNNBBBBSCCS] of the PUM2 binding element (PBE) is related to, but distinct from, the 3′ end of the Drosophila Nanos response element. The characterization of PUM2 and potential RNA-binding site will assist efforts to assess the extent and mechanism by which mammalian genes are regulated at a posttranscriptional level.