decay in Leishmania is coupled to translation
Résumé
Nous avons mis en évidence précédemment que des rétroposons éteints de la famille de SIDER2 (Short Interspersed DEgenarate Retroposons), situés majoritairement dans la région 3ʹUTR des transcrits chez Leishmania, favorisent la dégradation rapide des ARNm par clivage endonucléolytique. Dans cette étude, nous avons cherché à determiner si la dégradation des ARNm par SIDER2 est liée à la traduction. On démontre que l’inhibition de l’initiation de la traduction par l’insertion d’une structure en boucle dans la région 5ʹ d’un ARNm ayant une séquence SIDER2 dans le 3ʹUTR, a permis de bloquer la dégradation de l’ARNm. De même, l’inhibition de la traduction au niveau de la phase d’élongation par un traitement au cycloheximide inhibe la dégradation de l’ARNm de manière spécifique à SIDER2. Ces résultats suggèrent fortement que la dégradation des ARNm contenant SIDER2 est liée à la traduction, probablement via le recrutementde facteurs de dégradationau niveau des ribosomes actifs.
95
Succinctus
SIDER2 retroposon-mediated mRNA decay in
Leishmania is coupled to translation
Hiva Azizi1,2, Michaela Müller-McNicoll3 and Barbara Papadopoulou1,2* 1
Research Center in Infectious Diseases, CHU de Quebec Research Center, 2705 Laurier Blvd., Quebec, QC, Canada, G1V 4G2
2
Department of Microbiology-Infectious Diseases and Immunology, Faculty of Medicine, University Laval, Quebec, Canada
3
Institute for Cell Biology and Neuroscience, Goethe University Frankfurt, Max-von- Laue-Str. 13,60438 Frankfurt am Main, Frankfurt, Germany
*Corresponding author: Barbara Papadopoulou
CHU de Quebec Research Center, CHUL
2705 Laurier Blvd., Quebec (QC), Canada G1V 4G2 Phone: (418) 525-4444, ext. 47608; Fax: (418) 654-2715 Email: barbara.papadopoulou@crchul.ulaval.ca
96
Abstract
We have previously reported that Short Interspersed Degenerate Retroposons of the SIDER2 subfamily predominantly located within 3'UTRs of Leishmania transcripts promote rapid turnover that is initiated by endonucleolytic cleavage. Here, we investigated whether SIDER2-mediated decay is linked to translation. We show that inhibition of translation initiation by inserting a stem-loop structure in the 5'-end of a SIDER2-bearing transcript blocks mRNA degradation. Similarly, inhibition of translation elongation by cycloheximide leads to mRNA accumulation, which is dependent on SIDER2. Taken together, these findings support that degradation of SIDER2-harboring mRNAs takes place co-translationally, possibly through the recruitment of decay factors to elongating ribosomes.
Keywords: Leishmania; SIDER2 retroposon; mRNA decay; translation; MS2
97
mRNA turnover is an essential cellular process that determines the lifespan of cytoplasmic mRNAs, serving as a key regulator of gene expression at the post- transcriptional level under various conditions of growth. Particularly in Leishmania and related trypanosomatids, regulation of mRNA decay is of crucial importance as these organisms lack control at the level of transcription initiation and regulation of gene expression occurs exclusively at the post-transcriptional level (Haile and Papadopoulou, 2007). We have previously reported that Short Interspersed Degenerate Retroposons (SIDERs) of the SIDER2 subfamily in Leishmania promote rapid mRNA decay (Bringaud et al., 2007). SIDERs are truncated versions (~0.55 kb) of the formerly active ingi/L1Tc retroposons, predominantly located (>75%) in 3' untranslated regions (UTRs), with >20% of the Leishmania transcripts harboring SIDER elements (Bringaud et al., 2007; Smith et al., 2009). SIDER2-containing transcripts are generally short-lived and decay is initiated by endonucleolytic cleavage, for which the endonuclease is yet to be identified, without prior deadenylation (Müller et al., 2010a, b), the default process in eukaryotic mRNA decay (Parker, 2012; Roy and Jacobson, 2013). Cleavage of SIDER2-harboring transcripts was mapped within the second tandem 79 nucleotide (nt) hallmark sequence (Signature II) (Müller et al., 2010b), the most conserved sequence among SIDER2 retroposons (Bringaud et al., 2007; Smith et al., 2009). Deletion of the whole SIDER2 or Signature II blocks endonucleolytic cleavage and results in mRNA stabilization (Müller et al., 2010b).
Degradation of mRNAs is initiated through endonuclease-mediated cleavage in bacteria while in eukaryotes the initial step of mRNA turnover involves generally poly(A) shortening at the 3' end by deadenylases (Radhakrishnan and Green, 2016). Deadenylation is followed by 5'-3' decapping, Xrn1-mediated exonucleolytic decay or 3'-5' processing by the exosome (Parker, 2012). Heterogeneity in RNA stability can be attributed to 3' UTR regulatory motifs and trans-acting interacting factors or to microRNAs and non-coding RNAs. The effects of these multiple factors on mRNA decay have, in many cases, been associated with the translation machinery (Radhakrishnan and Green, 2016). Initially, it was perceived that mRNA degradation is a subsequent step to translation and that in order for an mRNA to be degraded, it
98
has first to exit translation (Coller and Parker, 2005). Although it has been shown that mRNAs undergoing translation are protected from degradation as elongating ribosomes limit mRNA decapping (Parker, 2012; Roy and Jacobson, 2013), multiple lines of evidence suggest that mRNAs can be decapped and undergo 5'-3' exonucleolytic decay while associated with ribosomes (Hu et al., 2009). There is indeed increasing evidence in the literature from bacteria, to yeast and metazoans that degradation of aberrant but also of normal mRNAs is regulated by their translational state and associated ribosomes (Pelechano et al., 2015; Radhakrishnan and Green, 2016). Relationships between mRNA degradation and translating ribosomes provide a quality control mechanism that minimizes the production of abnormal or harmful proteins. In bacteria, translating ribosomes are one of the most important factors influencing the lifespan of mRNAs (Laalami et al., 2014). Similarly, in eukaryotes, when elongation is impaired, the aberrant translational state of the mRNA and associated ribosomes appears to trigger decay. For example, ribosomal recognition of defective mRNAs triggers "mRNA surveillance" pathways that target the mRNA for degradation (Shoemaker and Green, 2012). There are also cases where mRNA surveillance pathways are co-opted to link translation to decay on functional mRNA transcripts, similar to the example of actively translated upstream open reading frames (ORFs) (Gaba et al., 2005; Hurt et al., 2013). More generally, the stability of functional mRNAs appears to be dictated by overall rates of translation by the ribosome (Presnyak et al., 2015). In yeast, it was shown that decay can occur while mRNAs are associated with actively translating ribosomes, indicating that dissociation of ribosomes from mRNA is not a prerequisite for decay (Hu et al., 2009).
In Leishmania, nothing is known yet about co-translational decay mechanisms. So here, we asked the question whether SIDER2-mediated decay is linked or not to translation. In other words, we investigated if ongoing translation is a prerequisite for the rapid turnover of SIDER2-bearing transcripts. To address this question, we made use of the luciferase (LUC) gene reporter system. First, we generated a construct with two tandem MS2 hairpin sequences placed upstream of the start codon of the LUC gene fused to the Leishmania infantum LinJ.36.4000 3' UTR
99
harboring a SIDER2 element (Fig. 1A; 2MS2-LUC-4000 3' UTR) in order to prevent ribosomes from reaching the AUG initiator codon and start translation. It has been shown previously that the presence of stable MS2 hairpin structures before the initiator codon could block translation, while when positioned after the codon stop those did not interfere with translation (Aharon and Schneider, 1993). Therefore, a control construct with MS2 hairpin sequences positioned just after the LUC stop codon was also made (Fig. 1A; LUC-2MS2-4000 3' UTR). A similar strategy was used for the LinJ.36.3990 transcript, adjacent to LinJ.36.4000 on chromosome 36, whose 3' UTR does not harbor a SIDER2 element (Fig. 1A; 2MS2-LUC-3990 3' UTR and LUC-2MS2-3990 3' UTR), for comparison purposes. As an additional SIDER2(-) control, a construct expressing 2MS2-LUC mRNA was also generated (Fig. 1A). Western blot experiments using an anti-LUC-specific antibody demonstrated that the presence of the tandem MS2 stem-loop upstream of the LUC gene, but not after the
LUC stop codon, successfully blocked LUC mRNA translation, regardless of the 3'
UTR (Fig. 1B). The use of an MS2 hairpin to inhibit translation has also been reported in the related organism Trypanosoma brucei (Shi et al., 2005). However, in
T. brucei translation initiation was blocked upon binding of the MS2 coat protein
(CP) to its cognate RNA (only one MS2 hairpin was used). In our hands, co- expressing in L. infantum the vectors described in Fig. 1A, together with a plasmid expressing MS2-CP, had the same inhibitory effect on translation, indicating that the two MS2 hairpins were sufficient to block translation (data not shown). Next, we examined the effect of translation initiation blockade on LUC transcript abundance by northern blot hybridization. In contrast to the actively translated LUC-2MS2-4000 3' UTR transcript, preventing translation initiation in cis blocked degradation of the 2MS2-LUC-4000 3' UTR mRNA and increased its accumulation by 2.5-fold (Fig. 1C). On the other hand, translation initiation blockade of the LUC-3990 3'UTR mRNA lacking SIDER2 had no effect on mRNA abundance (Fig. 1C). Similarly, translation inhibition of the 2MS2-LUC mRNA lacking 3' UTR and SIDER2 sequences did not increase mRNA accumulation (Fig. 1C). Reduction in the rate of translation initiation caused by a stem-loop in the mRNA 5' UTR leads generally to accelerated mRNA degradation through deadenylation and subsequent decapping
100
(Parker, 2012). As a result of deadenylation, PABP-eIF4G interactions stabilizing the translation initiation complex (Parker, 2012) can be affected, rendering the mRNA ends more accessible to their respective decay factors. Interestingly, here we obtained the opposite results as SIDER2-containing transcripts were stabilized upon inhibition of translation initiation in cis. The fact that SIDER2-mediated decay is deadenylation- and possibly decapping-independent (Müller et al., 2010b) may partly explain these observations.
Considering that a stable hairpin structure upstream of the start codon generally inhibits translation by blocking the formation of the pre-initiation complex and that insertion of a tandem MS2 stem-loop in the 5' UTR of a SIDER2-containing reporter transcript similarly blocked translation but also mRNA decay, we hypothesized that for SIDER2-harboring mRNAs to be degraded they have to associate with the translation apparatus. It is possible that the putative endoribonuclease and associated decay factors are recruited to the ribosome and target SIDER2-harboring mRNAs for degradation co-translationally. The fact that SIDER2 elements located in the 3' UTR of >1000 Leishmania transcripts share a conserved 79 nt signature II sequence (Smith et al., 2009), which was shown previously to be essential for cleavage and degradation (Müller et al., 2010b), favors a model where the endoribonuclease is recruited to translating ribosomes to reach its multiple mRNA targets. Several protein factors recruited to the ribosomes have been shown to mediate degradation of mRNA. For example, it was shown that exonucleases are running into actively translating ribosomes to degrade mRNAs in yeast (Hu et al., 2009; Pelechano et al., 2015). Also in yeast, mRNAs that sediment deep in a polysome profile were partially degraded by the exonuclease Xrn1 (Hu et al., 2009). The DEAD-box DHH1 RNA helicase was shown to couple mRNA decay and translation by monitoring codon optimality (Radhakrishnan and Green, 2016). Polysomal ribonuclease 1 (PMR1) is another well-documented endonuclease, which acts on polysome-bound mRNAs (Schoenberg, 2011). Active translation is required for PMR1-mediated decay, because inserting a stem-loop into the 5′ UTR to block translation inhibited endonucleolytic cleavage of the mRNA (Schoenberg, 2011). Also, microRNAs are recruited to mRNA targets in the ribosomes through Argonaute
101
proteins that bind to machineries implicated in mRNA decay (Behm-Ansmant et al., 2006).
Translation elongation inhibitors such as cycloheximide (CHX) are utilized to arrest ribosome translocation and to freeze them in their positions. In mammalian cells, it was shown than brief treatment with such drugs causes an accumulation of ribosomes in the first five to 10 codons of all genes (Ingolia et al., 2012). To further elaborate on the interplay between SIDER2-mediated mRNA decay and translation,
L. infantum recombinant parasites expressing a LUC reporter gene fused to the
LinJ.36.4000 3' UTR (LUC-4000 3' UTR) were treated with CHX and LUC mRNA levels were evaluated by northern blot hybridization at various time points post- treatment. Already after 30-60 min of CHX treatment, an effect on LUC mRNA accumulation was seen (Fig. 2A), in line with the need of ongoing translation for decay. Steady state levels of the LUC-4000 3' UTR transcript were increased by 4.0- fold after 4 h of CHX treatment (Fig. 2A) at similar rates to those obtained when deleting SIDER2 from LinJ.36.4000 3' UTR (Müller et al., 2010a, b). In addition, we examined the effect of CHX on decay rates of the endogenous LinJ.36.4000 and LinJ.08.1220 transcripts, both harboring a SIDER2 element in their 3′ UTR. Similarly, both transcripts were stabilized showing a 2.9- and 3.7-fold increased accumulation, respectively, after 6 h of CHX treatment in comparison with the untreated parasites (Fig. 2B). Inhibition of translation elongation often leads to mRNA stabilization (Parker, 2012; Roy and Jacobson, 2013). It has been postulated that the effect of CHX treatment on mRNA decay is indirect through inhibition of mRNA decapping (Beelman and Parker, 1994). Stalling ribosomes with CHX are no longer available for translation and re-initiation. Therefore, following CHX treatment, SIDER2-containing transcripts are stalled but mostly unoccupied and are therefore not degraded. In turn, efficient degradation would suggest that SIDER2-containing mRNAs are well occupied by translating ribosomes that have to deliver the endoribonuclease to the 3' UTR.
Blocking translation of labile transcripts such as those harboring an AU-rich element (ARE) in their 3' UTR (e.g. GM-CSF, c-fos, MFA2) did not systematically
102
affect decay rates (Koeller et al., 1991; Aharon and Schneider, 1993; Beelman and Parker, 1994). It is believed that differential outcomes of blocking translation on decay rates are mainly attributed to the cis-acting elements located within the 3' UTR and their interactions with trans-acting factors (Parker, 2012; Roy and Jacobson, 2013). Thus, we next evaluated the effect of a SIDER2(+) versus a SIDER2(-) 3' UTR on mRNA decay rates upon translation inhibition with CHX. Plasmids expressing a reporter gene (LUC) under the control of a SIDER2-harboring 3' UTR or solely the SIDER2 element or a 3' UTR lacking SIDER2 sequences were used for these studies. Because we were also interested to expand our investigation on the interplay between SIDER2-mediated mRNA decay and translation to other Leishmania spp., we used the Leishmania major LmjF.08.1270 and LmjF.36.3810 genes, the orthologs of L.
infantum LinJ.08.1220 and LinJ.36.4000, respectively. Leishmania major
recombinant parasites expressing the LUC reporter gene fused downstream with either the LmjF.08.1270 or LmjF.36.3810 3' UTRs (LUC-1270 3' UTR; LUC-3810 3'UTR) or SIDER2 alone (LUC-1270 SIDER2; LUC-3810 SIDER2) or a truncated 3'
UTR in which SIDER2 was deleted (LUC-1270 3'UTRΔSIDER2; LUC-3810
3'UTRΔSIDER2) were treated with CHX and total RNA was purified at indicated
time points and subjected to northern blot hybridization (Fig. 3). An increased accumulation of the LUC-3810 3' UTR transcript was observed with time exposure to CHX (Fig. 3A) in agreement with the results obtained with the L. infantum orthologous transcript 3' UTR (Fig. 2A). Similarly, the second SIDER2-containing transcript, LUC-1270 3' UTR, showed an increased accumulation of 4.5-fold after CHX treatment (Fig. 3B). Interestingly, the LUC-3810 SIDER2 and LUC-1270 SIDER2 transcripts, regulated solely by SIDER2, showed similar levels of accumulation upon CHX treatment with transcripts harboring the full-length 3' UTRs (Figs. 3A, B), indicating the dependence on SIDER2 for co-translational decay. On the other hand, CHX treatment had no effect on the decay of the stabilized LUC-1270
3'UTRΔSIDER2 and LUC-3810 3'UTRΔSIDER2 transcripts lacking SIDER2 (Figs.
3A, B). Leishmania major parasites treated with CHX demonstrated an increased accumulation of the endogenous LmjF.08.1270 and LmjF.36.3810 SIDER2-
103
harboring transcripts by 5.4- and 4.6-fold, respectively (Fig. 3C), in line with our observations using the LUC reporter transcripts (Fig. 3A and 3B).
CHX blocks translation elongation by freezing ribosomes on translating mRNAs, which shifts mRNA towards the polysomal fraction and might decrease the amount of non-polysomal mRNAs available for decay. To further assess the role of ongoing translation on SIDER2-mediated decay, we tested another translation inhibitor, puromycin (PUR), which causes premature chain termination, therefore shifting the mRNA polysomal pool towards free ribosomal fractions. If ongoing translation is needed for SIDER2-mediated decay, PUR treatment should stabilize SIDER2-containing transcripts. To this end, L. major parasites were treated with 200 g/ml of PUR and stabilization of LmjF.08.1270 and LmjF.36.3810 SIDER2- containing transcripts was evaluated by northern blot hybridization at different time points post-treatment. We observed that both 1270 and 3810 mRNAs increase in levels upon PUR treatment by 3.5- and 4.8-fold, respectively (Fig. 3C), similar to the results obtained with CHX (Fig. 3C). We have previously shown that the orthologous transcripts LinJ.08.1220 and LinJ.36.4000 in L. infantum were also stabilized following PUR treatment (Müller et al., 2010a). Finally, to address whether mRNA stabilization with PUR is dependent on SIDER2, we used two transgenic L. major
lines expressing LUC-1270 3'UTR or LUC-1270 3'UTRΔSIDER2. Northern blot
hybridization showed that increased accumulation of LUC-1270 mRNA upon PUR treatment was observed only in the presence of SIDER2 (Fig. 3D), which further supports that inhibition of translation elongation leads to stabilization of SIDER2- containing transcripts.
In conclusion, here we showed that SIDER2-mediated decay is equally blocked if translation initiation is prevented by a hairpin structure at the 5' end of the mRNA, or if translation is globally inhibited by CHX or PUR. Combined, these findings indicate that ongoing translation is a prerequisite for the rapid turnover of SIDER2-containing mRNAs. Our results support a co-translational mechanism of SIDER2-mediated mRNA decay, possibly through the recruitment of the endoribonuclease and associated decay factors to elongating ribosomes. This model is
104
in line with a coordinated regulated decay process targeting multiple SIDER2- harboring Leishmania transcripts encoding proteins of diverse functions and whose subcellular localization is essentially different. Additional studies to identify the SIDER2-specific endoribonuclease would further increase our understanding of this novel mechanism of regulated mRNA decay in Leishmania.
Acknowledgements
HA was a recipient of a fellowship from the Canadian Institutes of Health Research (CIHR) Strategic Training Program STP-53924 and the Centre for Host-Parasite Interactions (CHPI) funded by the Fonds de Recherche du Québec-Nature et Technologies. This work was supported by the Canadian Institutes of Health Research (Grant MOP-12182 awarded to BP).
105
References
Aharon, T., Schneider, R.J., 1993. Selective destabilization of short-lived mRNAs with the granulocyte-macrophage colony-stimulating factor AU-rich 3' noncoding region is mediated by a cotranslational mechanism. Mol Cell Biol 13, 1971-1980.
Beelman, C.A., Parker, R., 1994. Differential effects of translational inhibition in cis and in trans on the decay of the unstable yeast MFA2 mRNA. J Biol Chem 269, 9687-9692.
Behm-Ansmant, I., Rehwinkel, J., Doerks, T., Stark, A., Bork, P., Izaurralde, E., 2006. mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes. Genes Dev 20, 1885- 1898.
Bringaud, F., Müller, M., Cerqueira, G.C., Smith, M., Rochette, A., El-Sayed, N.M.A., Papadopoulou, B., Ghedin, E., 2007. Members of a large retroposon family are determinants of post-transcriptional gene expression in Leishmania. PLoS Pathog 3, e136.
Coller, J., Parker, R., 2005. General translational repression by activators of mRNA decapping. Cell 122, 875-886.
Gaba, A., Jacobson, A., Sachs, M.S., 2005. Ribosome occupancy of the yeast CPA1 upstream open reading frame termination codon modulates nonsense- mediated mRNA decay. Mol Cell 20, 449-460.
Haile, S., Papadopoulou, B., 2007. Developmental regulation of gene expression in trypanosomatid parasitic protozoa. Curr Opin Microbiol 10, 569-577.
Hu, W., Sweet, T.J., Chamnongpol, S., Baker, K.E., Coller, J., 2009. Co-translational mRNA decay in Saccharomyces cerevisiae. Nature 461, 225-229.
Hurt, J.A., Robertson, A.D., Burge, C.B., 2013. Global analyses of UPF1 binding and function reveal expanded scope of nonsense-mediated mRNA decay. Genome Res 23, 1636-1650.
Ingolia, N.T., Brar, G.A., Rouskin, S., McGeachy, A.M., Weissman, J.S., 2012. The ribosome profiling strategy for monitoring translation in vivo by deep
106
sequencing of ribosome-protected mRNA fragments. Nature Protocols 7, 1534-1550.
Keryer-Bibens, C., Barreau, C., Osborne, H.B., 2008. Tethering of proteins to RNAs by bacteriophage proteins. Biol Cell 100, 125-138.
Koeller, D.M., Horowitz, J.A., Casey, J.L., Klausner, R.D., Harford, J.B., 1991. Translation and the stability of mRNAs encoding the transferrin receptor and c-fos. Proc Natl Acad Sci U S A 88, 7778-7782.
Laalami, S., Zig, L., Putzer, H., 2014. Initiation of mRNA decay in bacteria. Cell Mol Life Sci 71, 1799-1828.
Müller, M., Padmanabhan, P.K., Papadopoulou, B., 2010a. Selective inactivation of SIDER2 retroposon-mediated mRNA decay contributes to stage- and species- specific gene expression in Leishmania. Mol Microbiol 77, 471-491.
Müller, M., Padmanabhan, P.K., Rochette, A., Mukherjee, D., Smith, M., Dumas, C., Papadopoulou, B., 2010b. Rapid decay of unstable Leishmania mRNAs bearing a conserved retroposon signature 3'-UTR motif is initiated by a site- specific endonucleolytic cleavage without prior deadenylation. Nucleic Acids Res 38, 5867-5883.
Parker, R., 2012. RNA degradation in Saccharomyces cerevisae. Genetics 191, 671- 702.
Pelechano, V., Wei, W., Steinmetz, L.M., 2015. Widespread co-translational RNA decay reveals ribosome dynamics. Cell 161, 1400-1412.
Presnyak, V., Alhusaini, N., Chen, Y.H., Martin, S., Morris, N., Kline, N., Olson, S., Weinberg, D., Baker, K.E., Graveley, B.R., Coller, J., 2015. Codon optimality is a major determinant of mRNA stability. Cell 160, 1111-1124.
Radhakrishnan, A., Green, R., 2016. Connections Underlying Translation and mRNA Stability. J Mol Biol 428, 3558-3564.
Roy, B., Jacobson, A., 2013. The intimate relationships of mRNA decay and translation. Trends Genet 29, 691-699.
Schoenberg, D.R., 2011. Mechanisms of endonuclease-mediated mRNA decay. Wiley Interdisciplinary Reviews: RNA 2, 582-600.
107
Shi, H., Djikeng, A., Chamond, N., Ngô, H., Tschudi, C., Ullu, E., 2005. Repression