Poly(A)-Specific Ribonuclease (PARN-1) Function in Stage-Specific mRNA Turnover in Trypanosoma brucei

Abstract

Deadenylation is often the rate-limiting event in regulating the turnover of cellular mRNAs in eukaryotes. Removal of the poly(A) tail initiates mRNA degradation by one of several decay pathways, including deadenylation-dependent decapping, followed by 5 to 3 exonuclease decay or 3 to 5 exosome-mediated decay. In trypanosomatids, mRNA degradation is important in controlling the expression of differentially expressed genes. Genomic annotation studies have revealed several potential deadenylases. Poly(A)-specific RNase (PARN) is a key deadenylase involved in regulating gene expression in mammals, Xenopus oocytes, and higher plants. Trypanosomatids possess three different PARN genes, PARN-1, -2, and -3, each of which is expressed at the mRNA level in two life-cycle stages of the human parasite Trypanosoma brucei. Here we show that T. brucei PARN-1 is an active deadenylase. To determine the role of PARN-1 on mRNA stability in vivo, we overexpressed this protein and analyzed perturbations in mRNA steady-state levels as well as mRNA half-life. Interestingly, a subset of mRNAs was affected, including a family of mRNAs that encode stage-specific coat proteins. These data suggest that PARN-1 functions in stage-specific protein production. Regulation of gene expression in the protozoan parasiteTrypanosoma brucei allows the organism to adapt and survive during its life cycle in two very different environments, the mammalian bloodstream and the tsetse fly. Expression of numerous protein-coding genes is regulated posttranscriptionally, particularly at the level of mRNA stability (4,11,25). For example, differential mRNA stability accounts for the stagespecific expression of procyclins, hexose transporters, and phosphoglycerate kinases (6,20,27,28,53).In the well-studied Saccharomyces and mammalian systems, mRNA decay is a tightly controlled, multistep, and multipathway process. Various cis-acting elements, embedded in specific mRNAs, are recognized by RNA-binding proteins (7,52,55), which stabilize mRNAs or recruit RNases to carry out mRNA degradation and inhibit translation (8,34,39,47). The removal of the mRNA 3Ј poly(A) tail by 3Ј to 5Ј exoribonucleases (deadenylases) is often the rate-limiting step in mRNA degradation in vertebrates and thus a key point in regulation of mRNA turnover (19,40).A number of different deadenylases exist in eukaryotes, including poly(A)-specific RNase (PARN), the CCR4/CAF1/ NOT complex, and the PAN2/PAN3 complex (reviewed in reference 23). The specific role of each of these proteins remains largely unknown, although evidence suggests that each enzyme may recognize a particular set of mRNA substrates (46). PARN functions in the targeted degradation of specific mRNAs in humans, Xenopus, and higher plants (1,3,22,31,32,36,58). To date, no PARN-encoding genes have been characterized in any single-cell eukaryote (56). In humans, PARN initiates decay of mRNAs containing AU-rich elements or nonsense codons. In Xenopus, PARN regulates oocyte maturation, whereas in Arabidopsis, PARN regulates embryogen...

Depletion of poly(A)-specific ribonuclease (PARN) inhibits proliferation of human gastric cancer cells by blocking cell cycle progression

“…Previous functional studies have shown that PARN is involved in several crucial physiological processes such as the meiotic maturation of frog oocytes [49], embryogenesis and stress response in plants [50,51] and stage-specific protein production in Trypanosoma brucei [52]. As for the cellular processes, PARN can regulate cell motility in mouse myoblasts [33], maturation of mammalian H/ACA box snoRNA [53], miRNA biogenesis [54] and DNA damage response (DDR) [31,32,55].…”

The roles of 3′-exoribonucleases and the exosome in trypanosome mRNA degradation

“…For most mRNAs, removal of the poly(A) tail precedes degradation of the remainder of the transcript (Manful et al 2011). A typical mRNA degradation machinery is present: an exosome (Estévez et al 2001; a scavenger decapping enzyme (Banerjee et al 2009); an Xrn1 homolog, XRNA (Li et al 2006); PAN2/PAN3 (Schwede et al 2009); PARN (Milone et al 2004;Utter et al 2011); a CAF1/NOT complex composed of CAF1, CAF40, NOT1, NOT2, NOT3/5 (Schwede et al 2008); a CNOT11 homolog (Schwede et al 2008;Mauxion et al 2013); and CNOT10 (Färber et al 2013). Only a Dcp2-like decapping enzyme is missing (Schwede et al 2009).…”

Global identification of conserved post-transcriptional regulatory programs in trypanosomatids

“…Also, we found that transcripts that have at least one instance of CoRM1970 are overrepresented in poly(A) + mRNA content of T. brucei cells that overexpress poly(A)-specific ribonuclease 1 (25), suggesting that CoRM1970-containing transcripts are also protected against deadenylation activity of this enzyme (Figure 2A). This is in line with previous reports showing that ELAV-like proteins can simultaneously bind to the ARE and poly(A) tail (46), and thus possibly protect the poly(A) tail of ARE-containing transcripts.…”

“…The motif name along with the structure/sequence is shown on the left, with each column representing one expression data set. The letters in brackets correspond to the reference publications that describe each experiment: a: (23), b: (26), c: (25), d: (21), e: (22). ( B ) Trypanosoma brucei genes (blue dots) are mapped on the first two principal components of 22 previously published expression data sets (21–27).…”