Double-stranded RNA induces mRNA degradation in Trypanosoma brucei

Double-stranded RNA (dsRNA) recently has been shown to give rise to genetic interference in Caenorhabditis elegans and also is likely to be the basis for phenotypic cosuppression in plants in certain instances. While constructing a plasmid vector for transfection of trypanosome cells, we serendipitously discovered that in vivo expression of dsRNA of the α-tubulin mRNA 5′ untranslated region (5′ UTR) led to multinucleated cells with striking morphological alterations and a specific block of cytokinesis. Transfection of synthetic α-tubulin 5′ UTR dsRNA, but not of either strand individually, caused the same phenotype. On dsRNA transfection, tubulin mRNA, but not the corresponding pre-mRNA, was rapidly and specifically degraded, leading to a deficit of α-tubulin synthesis. The transfected cells were no longer capable of carrying out cytokinesis and eventually died. Analysis of cytoskeletal structures from these trypanosomes revealed defects in the microtubules of the flagellar axoneme and of the flagellar attachment zone, a complex cortical structure that we propose is essential for establishing the path of the cleavage furrow at cytokinesis. Last, dsRNA-mediated mRNA degradation is not restricted to α-tubulin mRNA but can be applied to other cellular mRNAs, thus establishing a powerful tool to genetically manipulate these important protozoan parasites.

Recognition of Yeast mRNAs as “Nonsense Containing” Leads to Both Inhibition of mRNA Translation and mRNA Degradation: Implications for the Control of mRNA Decapping

A critical step in the degradation of many eukaryotic mRNAs is a decapping reaction that exposes the transcript to 5′ to 3′ exonucleolytic degradation. The dual role of the cap structure as a target of mRNA degradation and as the site of assembly of translation initiation factors has led to the hypothesis that the rate of decapping would be specified by the status of the cap binding complex. This model makes the prediction that signals that promote mRNA decapping should also alter translation. To test this hypothesis, we examined the decapping triggered by premature termination codons to determine whether there is a down-regulation of translation when mRNAs were recognized as “nonsense containing.” We constructed an mRNA containing a premature stop codon in which we could measure the levels of both the mRNA and the polypeptide encoded upstream of the premature stop codon. Using this system, we analyzed the effects of premature stop codons on the levels of protein being produced per mRNA. In addition, by using alterations either in cis or in trans that inactivate different steps in the recognition and degradation of nonsense-containing mRNAs, we demonstrated that the recognition of a nonsense codon led to a decrease in the translational efficiency of the mRNA. These observations argue that the signal from a premature termination codon impinges on the translation machinery and suggest that decapping is a consequence of the change in translational status of the mRNA.

A microarray study of post-mortem mRNA degradation in mouse brain tissue

Background: Although there is evidence that post-mortem interval (PMI) is not a major contributor to reduced overall RNA integrity, it may differentially affect a subgroup of gene transcripts that are susceptible to PMI-related degradation. This would particularly have ramifications for microarray studies that include a broad spectrum of genes. Method: Brain tissue was removed from adult mice at 0, 6, 12, 18, 24,36 and 48 h post-mortem. RNA transcript abundance was measured by hybridising RNA from the zero time point with test RNA from each PMI time point, and differential gene expression was assessed using cDNA microarrays. Sequence and ontological analyses were performed on the group of RNA transcripts showing greater than two-fold reduction. Results: Increasing PMI was associated with decreased tissue pH and increased RNA degradation as indexed by 28S/18S ribosomal RNA ratio. Approximately 12% of mRNAs detected on the arrays displayed more than a two-fold decrease in abundance by 48 It post-mortem. An analysis of nucleotide composition provided evidence that transcripts with the AUUUA motif in the 3' untranslated region (3'UTR) were more susceptible to PMI-related RNA degradation, compared to transcripts not carrying the 3'UTR AUUUA motif. Consistent with this finding, ontological analysis showed transcription factors and elements to be over-represented in the group of transcripts susceptible to degradation. Conclusion: A subgroup of mammalian mRNA transcripts are particularly susceptible to PMI-related degradation, and as a group, they are more likely to carry the YUTR AUUUA motif. PMI should be controlled for in human and animal model post-mortem brain studies, particularly those including a broad spectrum of mRNA transcripts. (c) 2005 Elsevier B.V. All rights reserved.

mRNA metabolism: nonsense mediated mRNA decay as a tool for gene therapy and the role of human DIS3L2 in transcript degradation

Tese de mestrado em Biologia Humana e Ambiente, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2015

SMG6 mediated degradation of nonsense mRNA requires phosphorylation-independent interaction with the helicase domain of UPF1

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and eliminated by nonsense-mediated mRNA decay (NMD). NMD targeted mRNAs can be degraded by different routes that all involve phosphorylated UPF1 (P-UPF1) as a starting point. The endonuclease SMG6, which cleaves mRNA near the PTC, is one of three known NMD factors thought to be recruited to nonsense mRNAs by interaction with P-UPF1, leading to eventual mRNA degradation. By MS2-mediated tethering of SMG6 and mutants thereof to a reporter RNA combined with knockdowns of various NMD factors, we demonstrate that besides its endonucleolytic activity, SMG6 also requires UPF1 and SMG1 for inducing RNA decay. Our experiments revealed a phosphorylation-independent interaction between SMG6 and UPF1 that is important for SMG6-mediated mRNA decay and using yeast two hybrid assays, we mapped this interaction to the unique stalk region of the UPF1 helicase domain. This region of UPF1 is essential for SMG6-mediated reporter RNA decay and also for NMD. Our results postulate that besides recruiting SMG6 to its RNA substrates, UPF1 is also required to activate its endonuclease activity.

SMG6 mediated degradation of nonsense mRNA requires phosphorylation-independent interaction with the helicase domain of UPF1

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and eliminated by nonsense-mediated mRNA decay (NMD). NMD targeted mRNAs can be degraded by different routes that all involve phosphorylated UPF1 (P-UPF1) as a starting point. The endonuclease SMG6, which cleaves mRNA near the PTC, is one of three known NMD factors thought to be recruited to nonsense mRNAs by interaction with P-UPF1, leading to eventual mRNA degradation. By MS2-mediated tethering of SMG6 and mutants thereof to a reporter RNA combined with knockdowns of various NMD factors, we demonstrate that besides its endonucleolytic activity, SMG6 also requires UPF1 and SMG1 for inducing RNA decay. Our experiments revealed a phosphorylation-independent interaction between SMG6 and UPF1 that is important for SMG6-mediated mRNA decay and using yeast two hybrid assays, we mapped this interaction to the unique stalk region of the UPF1 helicase domain. This region of UPF1 is essential for SMG6-mediated reporter RNA decay and also for NMD. Our results postulate that besides recruiting SMG6 to its RNA substrates, UPF1 is also required to activate its endonuclease activity.

Addition of destabilizing poly(A)-rich sequences to endonuclease cleavage sites during the degradation of chloroplast mRNA

In this work, we report the posttranscriptional addition of poly(A)-rich sequences to mRNA in chloroplasts of higher plants. Several sites in the coding region and the mature end of spinach chloroplast psbA mRNA, which encodes the D1 protein of photosystem II, are detected as polyadenylylated sites. In eukaryotic cells, the addition of multiple adenosine residues to the 3′ end of nuclear RNA plays a key role in generating functional mRNAs and in regulating mRNA degradation. In bacteria, the adenylation of several RNAs greatly accelerates their decay. The poly(A) moiety in the chloroplast, in contrast to that in eukaryotic nuclear encoded and bacterial RNAs, is not a ribohomopolymer of adenosine residues, but clusters of adenosines bounded mostly by guanosines and rarely by cytidines and uridines; it may be as long as several hundred nucleotides. Further analysis of the initial steps of chloroplast psbA mRNA decay revealed specific endonuclease cleavage sites that perfectly matched the sites where poly(A)-rich sequences were added. Our results suggest a mechanism for the degradation of psbA mRNA in which endonucleolytic cleavages are followed by the addition of poly(A)-rich sequences to the upstream cleavage products, which target these RNAs for rapid decay.

The molecular basis for selective inhibition of unconventional mRNA splicing by an IRE1-binding small molecule

IRE1 couples endoplasmic reticulum unfolded protein load to RNA cleavage events that culminate in the sequence-specific splicing of the Xbp1 mRNA and in the regulated degradation of diverse membrane-bound mRNAs. We report on the identification of a small molecule inhibitor that attains its selectivity by forming an unusually stable Schiff base with lysine 907 in the IRE1 endonuclease domain, explained by solvent inaccessibility of the imine bond in the enzyme-inhibitor complex. The inhibitor (abbreviated 4μ8C) blocks substrate access to the active site of IRE1 and selectively inactivates both Xbp1 splicing and IRE1-mediated mRNA degradation. Surprisingly, inhibition of IRE1 endonuclease activity does not sensitize cells to the consequences of acute endoplasmic reticulum stress, but rather interferes with the expansion of secretory capacity. Thus, the chemical reactivity and sterics of a unique residue in the endonuclease active site of IRE1 can be exploited by selective inhibitors to interfere with protein secretion in pathological settings.

Nuclear gene silencing directs reception of long-distance mRNA silencing in Arabidopsis

In plants, silencing of mRNA can be transmitted from cell to cell and also over longer distances from roots to shoots. To investigate the long-distance mechanism, WT and mutant shoots were grafted onto roots silenced for an mRNA. We show that three genes involved in a chromatin silencing pathway, NRPD1a encoding RNA polymerase IVa, RNA-dependent RNA polymerase 2 (RDR2), and DICER-like 3 (DCL3), are required for reception of long-distance mRNA silencing in the shoot. A mutant representing a fourth gene in the pathway, argonaute4 (ago4), was also partially compromised in the reception of silencing. This pathway produces 24-nt siRNAs and resulted in decapped RNA, a known substrate for amplification of dsRNA by RDR6. Activation of silencing in grafted shoots depended on RDR6, but no 24-nt siRNAs were detected in mutant rdr6 shoots, indicating that RDR6 also plays a role in initial signal perception. After amplification of decapped transcripts, DCL4 and DCL2 act hierarchically as they do in antiviral resistance to produce 21- and 22-nt siRNAs, respectively, and these guide mRNA degradation. Several dcl genotypes were also tested for their capacity to transmit the mobile silencing signal from the rootstock. dcl1-8 and a dcl2 dcl3 dcl4 triple mutant are compromised in micro-RNA and siRNA biogenesis, respectively, but were unaffected in signal transmission. © 2007 by The National Academy of Sciences of the USA.

Posttranscriptional regulation of acute phase serum amyloid A2 expression by the 5'- and 3'-untranslated regions of its mRNA

Human acute-phase serum amyloid A protein (A-SAA) is a major acute phase reactant, the concentration of which increases dramatically as part of the body's early response to inflammation. A-SAA is the product of two almost identical genes, SAA1 and SAA2, which are induced by the pro-inflammatory cytokines, IL-1 and IL-6. In this study, we examine the roles played by the 5'- and 3'-untranslated regions (UTRs) of the SAA2 mRNA in regulating A-SAA2 expression. SAA2 promoter-driven luciferase reporter gene constructs carrying the SAA2 5'-UTR and/or 3'-UTR were transiently transfected into the HepG2 human hepatoma cell line. After induction of chimeric mRNA with IL-1beta and IL-6, the SAA2 5'- and 3'-UTRs were both able to posttranscriptionally modify the expression of the luciferase reporter. The SAA2 5'-UTR promotes efficient translation of the chimeric luciferase transcripts, whereas the SAA2 3'-UTR shares this property and also significantly accelerates the rate of reporter mRNA degradation. Our data strongly suggest that the SAA2 5'- and 3'-UTRs each play significant independent roles in the posttranscriptional regulation of A-SAA2 protein synthesis.

Post-Transcriptional Regulation of BCL2 mRNA by the RNA-Binding Protein ZFP36L1 in Malignant B Cells

The human ZFP36 zinc finger protein family consists of ZFP36, ZFP36L1, and ZFP36L2. These proteins regulate various cellular processes, including cell apoptosis, by binding to adenine uridine rich elements in the 3′ untranslated regions of sets of target mRNAs to promote their degradation. The pro-apoptotic and other functions of ZFP36 family members have been implicated in the pathogenesis of lymphoid malignancies. To identify candidate mRNAs that are targeted in the pro-apoptotic response by ZFP36L1, we reverse-engineered a gene regulatory network for all three ZFP36 family members using the ‘maximum information coefficient’ (MIC) for target gene inference on a large microarray gene expression dataset representing cells of diverse histological origin. Of the three inferred ZFP36L1 mRNA targets that were identified, we focussed on experimental validation of mRNA for the pro-survival protein, BCL2, as a target for ZFP36L1. RNA electrophoretic mobility shift assay experiments revealed that ZFP36L1 interacted with the BCL2 adenine uridine rich element. In murine BCL1 leukemia cells stably transduced with a ZFP36L1 ShRNA lentiviral construct, BCL2 mRNA degradation was significantly delayed compared to control lentiviral expressing cells and ZFP36L1 knockdown in different cell types (BCL1, ACHN, Ramos), resulted in increased levels of BCL2 mRNA levels compared to control cells. 3′ untranslated region luciferase reporter assays in HEK293T cells showed that wild type but not zinc finger mutant ZFP36L1 protein was able to downregulate a BCL2 construct containing the BCL2 adenine uridine rich element and removal of the adenine uridine rich core from the BCL2 3′ untranslated region in the reporter construct significantly reduced the ability of ZFP36L1 to mediate this effect. Taken together, our data are consistent with ZFP36L1 interacting with and mediating degradation of BCL2 mRNA as an important target through which ZFP36L1 mediates its pro-apoptotic effects in malignant B-cells.