A Novel Member of the RNase D Exoribonuclease Family Functions in Mitochondrial Guide RNA Metabolism in Trypanosoma brucei

Abstract

RNA turnover and RNA editing are essential for regulation of mitochondrial gene expression in Trypanosoma brucei. RNA turnover is controlled in part by RNA 3 adenylation and uridylation status, with trans-acting factors also impacting RNA homeostasis. However, little is known about the mitochondrial degradation machinery or its regulation in T. brucei. We have identified a mitochondrial exoribonuclease, TbRND, whose expression is highly up-regulated in the insect proliferative stage of the parasite. TbRND shares sequence similarity with RNase D family enzymes but differs from all reported members of this family in possessing a CCHC zinc finger domain. In vitro, TbRND exhibits 3 to 5 exoribonuclease activity, with specificity toward uridine homopolymers, including the 3 oligo(U) tails of guide RNAs (gRNAs) that provide the sequence information for RNA editing. Several lines of evidence generated from RNAimediated knockdown and overexpression cell lines indicate that TbRND functions in gRNA metabolism in vivo. First, TbRND depletion results in gRNA tails extended by 2-3 nucleotides on average. Second, overexpression of wild type but not catalytically inactive TbRND results in a substantial decrease in the total gRNA population and a consequent inhibition of RNA editing. The observed effects on the gRNA population are specific as rRNAs, which are also 3-uridylated, are unaffected by TbRND depletion or overexpression. Finally, we show that gRNA binding proteins co-purify with TbRND. In summary, TbRND is a novel 3 to 5 exoribonuclease that appears to have evolved a function highly specific to the mitochondrion of trypanosomes.Recognition of the importance of post-transcriptional processes has changed the way we look at gene regulation (1). RNA degradation that occurs in both substrate processing and turnover is an important aspect of post-transcriptional regulation in all organisms (1-7), and the factors controlling these processes are beginning to be elucidated. Increasingly, we find that 3Ј non-encoded tails serve as cis-acting elements in RNA stability regulation. Perhaps the most commonly reported examples of this are oligo(A) tails acting in both RNA stabilization and destabilization (5, 8 -14). However, oligo(U) tails have also been found to destabilize microRNAs, siRNAs, and decay intermediates and possibly act as quality control mechanisms (15-20). Non-encoded 3Ј tails can recruit and influence the activity of exoribonucleases, which along with endoribonucleases, supply the catalytic activity necessary for RNA degradation. Exoribonucleases are organized into a number of different families or classes (21). Some of these enzymes, such as RNase II, PNPase, and Xrn1, exhibit processive activity and participate in overall RNA turnover pathways, although each is a member of a different family. Others, like RNase D, a member of the DEDD class of exoribonucleases, have distributive activity and function in very specific regulatory roles (21-23). In organelles, regulation of RNA stability and the classes of exoribonuc...

Proteomic Analysis Reveals Diverse Classes of Arginine Methylproteins in Mitochondria of Trypanosomes

“…The specificity of the assay was confirmed by the absence of TbRGG1 and TbRGG2 from control immunoprecipitations using normal rabbit serum. Additionally, a mitochondrial protein for which 70% coverage by MS failed to reveal any methylargs, TbRND (20), was absent from any of the immunoprecipitates. These data confirm the identity of two methylproteins detected by MS, and further suggest that these proteins are substrates for both ADMA and SDMA as reported for some methylproteins in yeast and mammals (42)(43)(44).…”

“…Anti-Hsp70 was a kind gift from Jay Bangs. Antibodies against TbRGG2, TbRGG1, and TbRND were described previously (19,20).…”

Functional characterization of two paralogs that are novel RNA binding proteins influencing mitochondrial transcripts of Trypanosoma brucei

“…A mt poly(A) polymerase (KPAP1) is responsible for the former structure while KPAP1 and RET1 act in concert with two pentatricopeptide repeat (PPR) proteins to produce the latter, a feature that marks mature mRNAs for translation (Ryan and Read 2005;Etheridge et al 2008;Aphasizheva et al 2011). In contrast to such a plethora of polyuridylylation events, a novel RNase D exoribonuclease targets the gRNA poly(U) tail, perhaps serving to fine-tune this heterogeneous population of molecules (Zimmer et al 2011). A close homolog of the RECC endonucleases has been recently shown to process polycistronic minicircle transcripts to yield functional gRNAs of the appropriate size and has thus been dubbed mtRNA precursorprocessing endonuclease 1 (mRPN1) (Madina et al 2011).…”

Multifunctional G-Rich and RRM-Containing Domains of TbRGG2 Perform Separate yet Essential Functions in Trypanosome RNA Editing

“…Fluorescence microscopy was performed as described previously (24,69). Cells were grown for 1 day in the presence of 2.5 g/ml tetracycline.…”