Trypanosome CNOT10 is essential for the integrity of the NOT deadenylase complex and for degradation of many mRNAs

Abstract

The degradation of most eukaryotic mRNAs is initiated by removal of the poly(A) tail, and the major deadenylase activity is associated with the CCR4/CAF1/NOT complex (NOT complex). We here study the role of CNOT10, a protein that is found in human and trypanosome, but not in yeast, NOT complexes. Trypanosome (Tb) CNOT10 is essential for growth. TbCNOT10 interacted with the deadenylase TbCAF1 and the scaffold protein TbNOT1; TbCAF1 also interacted with TbNOT1 in a yeast two-hybrid assay. In both trypanosomes and human embryonic kidney cells, approximately half of CAF1 was associated with the NOT complex. Depletion of CNOT10 from human cells did not affect this association. In contrast, depletion of TbCNOT10 in trypanosomes caused a decrease in the level of TbNOT1, detachment of TbCAF1 from the complex and pronounced stabilization of most trypanosome mRNAs. Artificial tethering of TbCAF1 to a reporter mRNA in vivo resulted in mRNA degradation, and this was not affected by TbCNOT10 depletion. We conclude that in trypanosomes, TbCNOT10 may stabilize the interaction between TbCAF1 and the NOT complex. The results further suggest that TbCAF1 is only able to deadenylate mRNA in vivo if it is recruited to the mRNA through other NOT complex components.

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

“…1; Table 1). The strong inhibition of mRNA decay after CNOT10 RNAi was previously reported (Färber et al 2013) and is illustrated again in Table 1, Figure 1, and Supplemental Figure S1. Bulk mRNA degradation in RRP45 RNAi lines is discussed in the next section.…”

“…For the RNA-seq analyses, we used existing cell lines (Estévez et al 2001;Schwede et al 2008Schwede et al , 2009Färber et al 2013). All lines were checked for the effectiveness of RNAi, by Northern blotting and by ensuring that growth was inhibited within 48 h of RNAi induction (data not shown), and later, more accurately, by analysis of the RNA-seq results (Table 1).…”

“…Depletion of XRNA (Li et al 2006), PAN2 (Schwede et al 2009), or the exosome (Haile et al 2003) delayed the degradation of the highly unstable developmentally regulated mRNA encoding EP procyclin, or reporter mRNAs bearing its 3 ′ UTR; ACT (actinA) mRNA, which has a half-life at the median, was also stabilized by RNAi targeting PAN2, and the developmentally regulated unstable mRNA encoding pyruvate phosphate decarboxylase was stabilized by RRP45 RNAi. In contrast, depletion of CAF1 (Schwede et al 2008(Schwede et al , 2009 or CNOT10 (Färber et al 2013) affected all mRNAs examined. Detailed studies of EP mRNA degradation kinetics provided clear evidence for a very rapid, deadenylation-independent, and XRNA-dependent 5 ′ -3 ′ pathway, combined with a slower deadenylation-dependent component (Irmer and Clayton 2001;Haile et al 2008;Schwede et al 2009).…”

“…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).…”

“…RRP45 is an essential structural component of the exosome; its depletion results in complex disassembly and instability of other exosome subunits (Estévez et al 2001. Similarly, CNOT10 is required for the attachment of the deadenylase CAF1 to the CAF1/NOT complex (Färber et al 2013). The median mRNA half-life in bloodstream-form trypanosomes (the form that grows in mammals) is ∼15-20 min (Manful et al 2011).…”