Trigger mRNA Degradation Research Articles

Systematic study of cis-antisense miRNAs in animal species reveals miR-3661 to target PPP2CA in human cells.

MicroRNAs (miRNAs) suppress targeting gene expression through blocking translation or triggering mRNA degradation and, in general, act in trans, through a partially complementary interaction with the 3′ untranslated region (3′ UTR) or coding regions of a target gene. Although it has been reported previously that some miRNAs suppress their target genes on the opposite strand with a fully complementary sequence (i.e., natural antisense miRNAs that act in cis), there is no report to systematically study such cis-antisense miRNAs in different animal species. Here we report that cis-antisense miRNAs do exist in different animal species: 48 in Caenorhabditis elegans, 17 in Drosophila, 36 in Mus musculus, and 52 in Homo sapiens using a systematical bioinformatics approach. We show that most of these cis-antisense miRNAs can efficiently reduce the expression levels of their target genes in human cells. We further investigate hsa-miR-3661, one of the predicted cis-antisense miRNAs, in detail and demonstrate that this miRNA directly targets the coding sequence of PPP2CA located on the opposite DNA strand and inhibits the PPP2CA expression. Taken together, these results indicate that cis-antisense miRNAs are conservative and functional in animal species including humans.

A cytoplasmic pathway for gapmer antisense oligonucleotide-mediated gene silencing in mammalian cells.

Antisense oligonucleotides (ASOs) are known to trigger mRNA degradation in the nucleus via an RNase H-dependent mechanism. We have now identified a putative cytoplasmic mechanism through which ASO gapmers silence their targets when transfected or delivered gymnotically (i.e. in the absence of any transfection reagent). We have shown that the ASO gapmers can interact with the Ago-2 PAZ domain and can localize into GW-182 mRNA-degradation bodies (GW-bodies). The degradation products of the targeted mRNA, however, are not generated by Ago-2-directed cleavage. The apparent identification of a cytoplasmic pathway complements the previously known nuclear activity of ASOs and concurrently suggests that nuclear localization is not an absolute requirement for gene silencing.

Nonsense mutations in the rhodopsin gene that give rise to mild phenotypes trigger mRNA degradation in human cells by nonsense-mediated decay

Eight different nonsense mutations in the human rhodopsin gene cause retinitis pigmentosa (RP), an inherited degenerative disease of the retina that can lead to complete blindness. Although all these nonsense mutations lead to premature termination codons (PTCs) in rhodopsin mRNA, some display dominant inheritance, while others are recessive. Because nonsense-mediated decay (NMD) can degrade mRNAs containing PTCs and modulate the inheritance patterns of genetic diseases, we asked whether any of the nonsense mutations in the rhodopsin gene generated mRNAs that were susceptible to degradation by NMD. We hypothesized that nonsense mutations that caused mild RP phenotypes would trigger NMD, whereas those that did not engage NMD would cause more severe RP phenotypes—presumably due to the toxicity of the truncated protein. To test our hypothesis, we transfected human rhodopsin nonsense mutants into HEK293 and HT1080 human cells and measured transcript levels by qRT-PCR. In both cell lines, rhodopsin mutations Q64X and Q344X, which cause severe phenotypes that are dominantly inherited, yielded the same levels of rhodopsin mRNA as wild type. By contrast, rhodopsin mutations W161X and E249X, which cause recessive RP, showed decreased rhodopsin mRNA levels, consistent with NMD. Rhodopsin mutant Y136X, a dominant mutation that causes late-onset RP with a very mild pathology, also gave lower mRNA levels. Treatment of cells with Wortmannin, an inhibitor of NMD, eliminated the degradation of Y136X, W161X, and E249X rhodopsin mRNAs. These results suggest that NMD modulates the severity of RP in patients with nonsense mutations in the rhodopsin gene.

MicroRNAs: heralds of the noncoding RNA revolution.

MicroRNAs: heralds of the noncoding RNA revolution.

Regulation of splicing factors by alternative splicing and NMD is conserved between kingdoms yet evolutionarily flexible.

Ultraconserved elements, unusually long regions of perfect sequence identity, are found in genes encoding numerous RNA-binding proteins including arginine-serine rich (SR) splicing factors. Expression of these genes is regulated via alternative splicing of the ultraconserved regions to yield mRNAs that are degraded by nonsense-mediated mRNA decay (NMD), a process termed unproductive splicing (Lareau et al. 2007; Ni et al. 2007). As all human SR genes are affected by alternative splicing and NMD, one might expect this regulation to have originated in an early SR gene and persisted as duplications expanded the SR family. But in fact, unproductive splicing of most human SR genes arose independently (Lareau et al. 2007). This paradox led us to investigate the origin and proliferation of unproductive splicing in SR genes. We demonstrate that unproductive splicing of the splicing factor SRSF5 (SRp40) is conserved among all animals and even observed in fungi; this is a rare example of alternative splicing conserved between kingdoms, yet its effect is to trigger mRNA degradation. As the gene duplicated, the ancient unproductive splicing was lost in paralogs, and distinct unproductive splicing evolved rapidly and repeatedly to take its place. SR genes have consistently employed unproductive splicing, and while it is exceptionally conserved in some of these genes, turnover in specific events among paralogs shows flexible means to the same regulatory end.

Transcription-coupled RNA surveillance in human genetic diseases caused by splice site mutations.

Current estimates indicate that approximately one-third of all disease-causing mutations are expected to disrupt splicing. Abnormal splicing often leads to disruption of the reading frame with introduction of a premature termination codon (PTC) that targets the mRNA for degradation in the cytoplasm by nonsense mediated decay (NMD). In addition to NMD there are RNA surveillance mechanisms that act in the nucleus while transcripts are still associated with the chromatin template. However, the significance of nuclear RNA quality control in the context of human genetic diseases is unknown. Here we used patient-derived lymphoblastoid cell lines as disease models to address how biogenesis of mRNAs is affected by splice site mutations. We observed that most of the mutations analyzed introduce PTCs and trigger mRNA degradation in the cytoplasm. However, for some mutant transcripts, RNA levels associated with chromatin were found down-regulated. Quantification of nascent transcripts further revealed that a subset of genes containing splicing mutations (SM) have reduced transcriptional activity. Following treatment with the translation inhibitor cycloheximide the cytoplasmic levels of mutant RNAs increased, while the levels of chromatin-associated transcripts remained unaltered. These results suggest that transcription-coupled surveillance mechanisms operate independently from NMD to reduce cellular levels of abnormal RNAs caused by SM.

Regulation of CTR2 mRNA by the nonsense-mediated mRNA decay pathway

The nonsense-mediated mRNA decay (NMD) pathway was originally identified as a pathway that degrades mRNAs with premature termination codons; however, NMD is now known to regulate natural mRNAs as well. Natural mRNAs are degraded by NMD due to the presence of specific NMD targeting features. An atypically long 3′-UTR is one of the features that has been shown to induce the rapid degradation of mRNAs by NMD in Saccharomyces cerevisiae and other organisms. S. cerevisiae CTR2 mRNAs have long 3′-UTRs and are sensitive to NMD, although the extent by which these long 3′-UTRs target the CTR2 mRNAs to the pathway is unknown. Here, we investigated the sequence elements that induce NMD of the CTR2 mRNAs and determined that the long CTR2 3′-UTR is sufficient to target an NMD-insensitive mRNA to the pathway. We also found that, although the CTR2 3′-UTR contributes to NMD-induced degradation, CTR2 mRNAs contain additional NMD-inducing features that function cooperatively with the atypically long 3′-UTR to trigger mRNA degradation. Lengthening the CTR2 ORF abrogates NMD and renders the mRNAs immune to the NMD pathway. Moreover, we found that transcription of CTR2 driven by the GPD promoter, which is not identical to the CTR2 promoter, affects degradation of the transcripts by NMD.

The interaction of cytoplasmic poly(A)-binding protein with eukaryotic initiation factor 4G suppresses nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) eliminates different classes of mRNA substrates including transcripts with long 3′ UTRs. Current models of NMD suggest that the long physical distance between the poly(A) tail and the termination codon reduces the interaction between cytoplasmic poly(A)-binding protein (PABPC1) and the eukaryotic release factor 3a (eRF3a) during translation termination. In the absence of PABPC1 binding, eRF3a recruits the NMD factor UPF1 to the terminating ribosome, triggering mRNA degradation. Here, we have used the MS2 tethering system to investigate the suppression of NMD by PABPC1. We show that tethering of PABPC1 between the termination codon and a long 3′ UTR specifically inhibits NMD-mediated mRNA degradation. Contrary to the current model, tethered PABPC1 mutants unable to interact with eRF3a still efficiently suppress NMD. We find that the interaction of PABPC1 with eukaryotic initiation factor 4G (eIF4G), which mediates the circularization of mRNAs, is essential for NMD inhibition by tethered PABPC1. Furthermore, recruiting either eRF3a or eIF4G in proximity to an upstream termination codon antagonizes NMD. While tethering of an eRF3a mutant unable to interact with PABPC1 fails to suppress NMD, tethered eIF4G inhibits NMD in a PABPC1-independent manner, indicating a sequential arrangement of NMD antagonizing factors. In conclusion, our results establish a previously unrecognized link between translation termination, mRNA circularization, and NMD suppression, thereby suggesting a revised model for the activation of NMD at termination codons upstream of long 3′ UTR.

Analysis of CDS-located miRNA target sites suggests that they can effectively inhibit translation

Most of what is presently known about how miRNAs regulate gene expression comes from studies that characterized the regulatory effect of miRNA binding sites located in the 3′ untranslated regions (UTR) of mRNAs. In recent years, there has been increasing evidence that miRNAs also bind in the coding region (CDS), but the implication of these interactions remains obscure because they have a smaller impact on mRNA stability compared with miRNA-target interactions that involve 3′ UTRs. Here we show that miRNA-complementary sites that are located in both CDS and 3′-UTRs are under selection pressure and share the same sequence and structure properties. Analyzing recently published data of ribosome-protected fragment profiles upon miRNA transfection from the perspective of the location of miRNA-complementary sites, we find that sites located in the CDS are most potent in inhibiting translation, while sites located in the 3′ UTR are more efficient at triggering mRNA degradation. Our study suggests that miRNAs may combine targeting of CDS and 3′ UTR to flexibly tune the time scale and magnitude of their post-transcriptional regulatory effects.

The 2',3'-cAMP-adenosine pathway.

Our recent studies employing HPLC-tandem mass spectrometry to analyze venous perfusate from isolated, perfused kidneys demonstrate that intact kidneys produce and release into the extracellular compartment 2',3'-cAMP, a positional isomer of the second messenger 3',5'-cAMP. To our knowledge, this represents the first detection of 2',3'-cAMP in any cell/tissue/organ/organism. Nuclear magnetic resonance experiments with isolated RNases and experiments in isolated, perfused kidneys suggest that 2',3'-cAMP likely arises from RNase-mediated transphosphorylation of mRNA. Both in vitro and in vivo kidney experiments demonstrate that extracellular 2',3'-cAMP is efficiently metabolized to 2'-AMP and 3'-AMP, both of which can be further metabolized to adenosine. This sequence of reactions is called the 2',3'-cAMP-adenosine pathway (2',3'-cAMP → 2'-AMP/3'-AMP → adenosine). Experiments in rat and mouse kidneys show that metabolic poisons increase extracellular levels of 2',3'-cAMP, 2'-AMP, 3'-AMP, and adenosine; however, little is known regarding the pharmacology of 2',3'-cAMP, 2'-AMP, and 3'-AMP. What is known is that 2',3'-cAMP facilitates activation of mitochondrial permeability transition pores, a process that can lead to apoptosis and necrosis, and inhibits proliferation of vascular smooth muscle cells and glomerular mesangial cells. In summary, there is mounting evidence that at least some types of cellular injury, by triggering mRNA degradation, engage the 2',3'-cAMP-adenosine pathway, and therefore this pathway should be added to the list of biochemical pathways that produce adenosine. Although speculative, it is possible that the 2',3'-cAMP-adenosine pathway may protect against some forms of acute organ injury, for example acute kidney injury, by both removing an intracellular toxin (2',3'-cAMP) and increasing an extracellular renoprotectant (adenosine).

Expression levels of microRNAs are not associated with their regulatory activities

MicroRNAs (miRNAs) regulate their targets by triggering mRNA degradation or translational repression. The negative relationship between miRNAs and their targets suggests that the regulatory effect of a miRNA could be determined from the expression levels of its targets. Here, we investigated the relationship between miRNA activities determined by computational programs and miRNA expression levels by using data in which both mRNA and miRNA expression from the same samples were measured. We found that different from the intuitive expectation one might have, miRNA activity shows very weak correlation with miRNA expression, which indicates complex regulating mechanisms between miRNAs and their target genes.ReviewersThis manuscript was reviewed by an anonymous reviewer and Dr Yuriy Gusev.

TRAP150 activates pre-mRNA splicing and promotes nuclear mRNA degradation

TRAP150 has been identified as a subunit of the transcription regulatory complex TRAP/Mediator, and also a component of the spliceosome. The exact function of TRAP150, however, remains unclear. We recently identified TRAP150 by its association with the mRNA export factor TAP. TRAP150 contains an arginine/serine-rich domain and has sequence similarity with the cell death-promoting transcriptional repressor BCLAF1. We found that TRAP150 co-localizes with splicing factors in nuclear speckles, and is required for pre-mRNA splicing and activates splicing in vivo. TRAP150 remains associated with the spliced mRNA after splicing, and accordingly, it interacts with the integral exon junction complex. Unexpectedly, when tethered to a precursor mRNA, TRAP150 can trigger mRNA degradation in the nucleus. However, unlike nonsense-mediated decay, TRAP150-mediated mRNA decay is irrespective of the presence of upstream stop codons and occurs in the nucleus. Moreover, TRAP150 activates pre-mRNA splicing and induces mRNA degradation by its separable functional domains. Therefore, TRAP150 represents a multi-functional protein involved in nuclear mRNA metabolism.

Relative contribution of sequence and structure features to the mRNA binding of Argonaute/EIF2C–miRNA complexes and the degradation of miRNA targets

How miRNAs recognize their target sites is a puzzle that many experimental and computational studies aimed to solve. Several features, such as perfect pairing of the miRNA seed, additional pairing in the 3' region of the miRNA, relative position in the 3' UTR, and the A/U content of the environment of the putative site, have been found to be relevant. Here we have used a large number of previously published data sets to assess the power that various sequence and structure features have in distinguishing between putative sites that do and those that do not appear to be functional. We found that although different data sets give widely different answers when it comes to ranking the relative importance of these features, the sites inferred from most transcriptomics experiments, as well as from comparative genomics, appear similar at this level. This suggests that miRNA target sites have been selected in evolution on their ability to trigger mRNA degradation. To understand at what step in the miRNA-induced response individual features play a role, we transfected human HEK293 cells with miRNAs and analyzed the association of Argonaute/EIF2C-miRNA complexes with target mRNAs and the degradation of these messages. We found that structural features of the target site are only important for Argonaute/EIF2C binding, while sequence features such as the A/U content of the 3' UTR are important for mRNA degradation.

1H, 13C and 15N resonance assignments of RNA pyrophosphohydrolase RppH from Escherichia coli

The mRNA degradation is an important regulatory mechanism which controls gene expression by limiting the number of translation times. Previous studies demonstrated that this process is essential for organisms. Escherichia coli RNA pyrophosphohydrolase (RppH) is an enzyme that triggers mRNA degradation by removing the 5' pyrophosphate, which is a rate-determining step. In order to understand the molecular mechanism of the biological function, the structural information of RppH is required. Herein, we report the resonance assignments of (1)H, (15)N, (13)C atoms of the E. coli RppH.

Two Molecular Pathways (NMD and ERAD) Contribute to a Genetic Epilepsy Associated with the GABAAReceptor GABRA1 PTC Mutation, 975delC, S326fs328X

Approximately one-third of human genetic diseases are caused by premature translation-termination codon (PTC)-generating mutations. These mutations in sodium channel and GABA(A) receptor genes have been associated with idiopathic generalized epilepsies, but the cellular consequences of the PTCs on the mutant channel subunit biogenesis and function are unknown. The PTCs could result in translation of a truncated subunit, or more likely, trigger mRNA degradation through nonsense-mediated mRNA decay (NMD), thus preventing or reducing production of mutant subunit at the transcriptional level. The GABA(A) receptor alpha1 subunit mutation, 975delC, S326fs328X, is an autosomal dominant mutation associated with childhood absence epilepsy that generates a PTC in exon 8 of the 9 exon GABRA1 gene that is 74 bp upstream of intron 8. Using an intron 8-inclusion minigene that supports NMD, we demonstrated that mutant mRNA was substantially reduced, but not absent. Loss of mutant transcripts was blocked by ribosome inhibition or by silencing the NMD-essential gene hUPF-1. In both neurons and non-neuronal cells, the PTC caused substantial loss of mutant alpha1(S326fs328X) subunit mRNA through NMD with a minor portion of the mRNA escaping NMD and producing a mutant protein. The translated mutant protein had reduced stability due to endoplasmic reticulum associated degradation (ERAD) and had enhanced association with molecular chaperones. This study suggests that loss of mRNA due to activation of NMD and activation of ERAD by the mutant protein may contribute to epileptogenesis. The molecular mechanisms outlined here delineate a model for the pathogenesis of many PTC-generating mutations.

Deadenylation is a widespread effect of miRNA regulation

miRNAs silence gene expression by repressing translation and/or by promoting mRNA decay. In animal cells, degradation of partially complementary miRNA targets occurs via deadenylation by the CAF1-CCR4-NOT1 deadenylase complex, followed by decapping and subsequent exonucleolytic digestion. To determine how generally miRNAs trigger deadenylation, we compared mRNA expression profiles in D. melanogaster cells depleted of AGO1, CAF1, or NOT1. We show that approximately 60% of AGO1 targets are regulated by CAF1 and/or NOT1, indicating that deadenylation is a widespread effect of miRNA regulation. However, neither a poly(A) tail nor mRNA circularization are required for silencing, because mRNAs whose 3' ends are generated by a self-cleaving ribozyme are also silenced in vivo. We show further that miRNAs trigger mRNA degradation, even when binding by 40S ribosomal subunits is inhibited in cis. These results indicate that miRNAs promote mRNA decay by altering mRNP composition and/or conformation, rather than by directly interfering with the binding and function of ribosomal subunits.

Targeting of Aberrant mRNAs to Cytoplasmic Processing Bodies

In eukaryotes, a specialized pathway of mRNA degradation termed nonsense-mediated decay (NMD) functions in mRNA quality control by recognizing and degrading mRNAs with aberrant termination codons. We demonstrate that NMD in yeast targets premature termination codon (PTC)-containing mRNA to P-bodies. Upf1p is sufficient for targeting mRNAs to P-bodies, whereas Upf2p and Upf3p act, at least in part, downstream of P-body targeting to trigger decapping. The ATPase activity of Upf1p is required for NMD after the targeting of mRNAs to P-bodies. Moreover, Upf1p can target normal mRNAs to P-bodies but not promote their degradation. These observations lead us to propose a new model for NMD wherein two successive steps are used to distinguish normal and aberrant mRNAs.

SiRNA-mediated off-target gene silencing triggered by a 7 nt complementation

A growing body of evidence suggests that siRNA could generate off-target effects through different mechanisms. However, the full impact of off-target gene regulation on phenotypic induction and accordingly on data interpretation in the context of large-scale siRNA library screen has not been reported. Here we report on off-target gene silencing effects observed in a large-scale knockdown experiment designed to identify novel regulators of the HIF-1 pathway. All of the three ‘top hits’ from our screen have been demonstrated to result from off-target gene silencing. Two of the three ‘siRNA hits’ were found to directly trigger down-regulation of hif-1α mRNA through a 7 nt motif, AGGCAGT, that is present in both the hif-1α mRNA and the siRNAs. Further analysis revealed that the generation of off-target gene silencing via this 7 nt motif depends on the characteristics of the target mRNA, including the sequence context surrounding the complementary region, the position of the complementary region in the mRNA and the copy number of the complementary region. Interestingly, the off-target siRNA against hif-1α was also shown to trigger mRNA degradation with high probability of other genes that possess multiple copies of the AGGCAGT motif in the 3′-untranslated region. Lessons learned from this study will be a valuable asset to aid in designing siRNAs with more stringent target selectivity and improving ‘hits-follow-up’ strategies for future large-scale knockdown experiments.

Degradation of targeted mRNAs in Escherichia coli: regulation by a small antisense RNA.

In this issue of Genes & Development, Masse et al. (2003a) show that a small antisense RNA (saRNA) triggers mRNA degradation via a pathway in which the saRNA is also consumed. The stoichiometric binding of the saRNA to its mRNA target thus appears to activate the degradation of both RNAs (coupled degradation). The physical interaction of complementary polynucleotides is arguably the fundamental chemical principle underlying molecular genetics. It is the most important feature of the structure of double-stranded DNA, and it is at the heart of the mechanism by which tRNA decodes mRNA to synthesize protein. The article by Masse et al. (2003a) reports novel findings on the action of a chromosome-encoded saRNA in the regulation of gene expression. They show that RyhB, an saRNA, triggers mRNA degradation via a pathway in which RyhB is degraded together with its target mRNA. Similar results were obtained with two other saRNAs. Thus, it seems likely that RyhB will serve as a model for saRNAs that activate mRNA degradation. Biological regulatory mechanisms involving antisense RNA have been recognized for many years in Escherichia coli. However, until recently, the known antisense RNAs were encoded by extrachromosomal elements such as transposons, plasmids, and bacteriophage. With the current renaissance in interest in small noncoding RNAs, a surprising number of putative small regulatory RNAs (50–100), encoded on the E. coli chromosome, have recently been described. An important factor in the discovery of these RNAs was the availability of the complete genome sequence of E. coli and related bacteria such as Salmonella. This genomic information facilitated searches based, in part, on sequence conservation in noncoding regions of the chromosome. It is only now that the function of some of the newly discovered RNAs is being elucidated. Although it seems reasonable to believe that the chromosome-encoded saRNAs and the classical antisense RNAs from transposons, plasmids, and bacteriophage act by similar mechanisms, I will argue that there could be some significant differences. This perspective is based on the accompanying research article by Masse et al. (2003a). As a reference for recent developments on small regulatory RNAs in E. coli, I highly recommend the review by Susan Gottesman that was published in Genes & Development last year (Gottesman 2002). In this perspective, I will describe the antisense RNAs from transposons, plasmids, and bacteriophage that have been characterized over the past twenty years (classical antisense), then introduce the chromosome-encoded antisense RNAs that have been discovered recently (renaissance antisense). After that, I will briefly discuss some differences between these two families of antisense RNA, then discuss the work in the Masse et al. (2003a) article and its implications for the role of the chromosome-encoded antisense RNAs in mRNA degradation.