The Upf-dependent decay of wild-type PPR1 mRNA depends on its 5'-UTR and first 92 ORF nucleotides

mRNAs containing premature translation termination codons (nonsense mRNAs) are targeted for deadenylation-independent degradation in a mechanism that depends on Upf1p, Upf2p and Upf3p. This decay pathway is often called nonsense- mediated mRNA decay (NMD). Nonsense mRNAs are decapped by Dcp1p and then degraded 5′ to 3′ by Xrn1p. In the yeast Saccharomyces cerevisiae, a significant number of wild-type mRNAs accumulate in upf mutants. Wild-type PPR1 mRNA is one of these mRNAs. Here we show that PPR1 mRNA degradation depends on the Upf proteins, Dcp1p, Xrn1p and Hrp1p. We have mapped an Upf1p-dependent destabilizing element to a region located within the 5′-UTR and the first 92 bases of the PPR1 ORF. This element targets PPR1 mRNA for Upf-dependent decay by a novel mechanism.     

Identification of elements in human long 3′ UTRs that inhibit nonsense-mediated decay

The nonsense-mediated mRNA decay (NMD) pathway serves an important role in gene expression by targeting aberrant mRNAs that have acquired premature termination codons (PTCs) as well as a subset of normally processed endogenous mRNAs. One determinant for the targeting of mRNAs by NMD is the occurrence of translation termination distal to the poly(A) tail. Yet, a large subset of naturally occurring mRNAs contain long 3′ UTRs, many of which, according to global studies, are insensitive to NMD. This raises the possibility that such mRNAs have evolved mechanisms for NMD evasion. Here, we analyzed a set of human long 3′ UTR mRNAs and found that many are indeed resistant to NMD. By dissecting the 3′ UTR of one such mRNA, TRAM1 mRNA, we identified a cis element located within the first 200 nt that inhibits NMD when positioned in downstream proximity of the translation termination codon and is sufficient for repressing NMD of a heterologous reporter mRNA. Investigation of other NMD-evading long 3′ UTR mRNAs revealed a subset that, similar to TRAM1 mRNA, contains NMD-inhibiting cis elements in the first 200 nt. A smaller subset of long 3′ UTR mRNAs evades NMD by a different mechanism that appears to be independent of a termination-proximal cis element. Our study suggests that different mechanisms have evolved to ensure NMD evasion of human mRNAs with long 3′ UTRs.     

AUUUA Sequences Direct mRNA Deadenylation Uncoupled from Decay during Xenopus Early Development

To study the regulation of AUUUA-mediated RNA deadenylation and destabilization during Xenopus early development, we microinjected chimeric mRNAs containing Xenopus or mammalian 3′ untranslated region (3′-UTR) sequences into Xenopus oocytes, mature eggs, or fertilized embryos. We found that the AU-rich elements (ARE) of Xenopus c-myc II and the human granulocyte-macrophage colony-stimulating factor gene (GMCSF) both direct deadenylation of chimeric mRNAs in an AUUUA-dependent manner. In the case of the Xenopus c-myc II ARE, mutation of a single AUUUA within an absolutely conserved 11-nucleotide region in c-myc 3′-UTRs prevents ARE-mediated deadenylation. AUUUA-specific deadenylation appears to be developmentally regulated: low deadenylation activity is observed in the oocyte, whereas rapid deadenylation occurs following egg activation or fertilization. Deadenylation results in the accumulation of stable deadenylated RNAs that become degraded only following mid-blastula transition. We conclude that ARE-mediated mRNA deadenylation can be uncoupled from ARE-mediated mRNA decay and that AUUUAs directly signal deadenylation during Xenopus early development.     

Identification and characterization of extensive intra-molecular associations between 3'-UTRs and their ORFs

During eukaryotic translation, mRNAs may form intra-molecular interactions between distant domains. The 5′-cap and the polyA tail were shown to interact through their associated proteins, and this can induce physical compaction of the mRNA in vitro. However, the stability of this intra-molecular association in translating mRNAs and whether additional contacts exist in vivo are largely unknown. To explore this, we applied a novel approach in which several endogenous polysomal mRNAs from Saccharomyces cerevisiae were cleaved near their stop codon and the resulting 3′-UTR fragments were tested either for co-sedimentation or co-immunoprecipitation (co-IP) with their ORFs. In all cases a significant fraction of the 3′-UTR fragments sedimented similarly to their ORF-containing fragments, yet the extent of co-sedimentation differed between mRNAs. Similar observations were obtained by a co-IP assay. Interestingly, various treatments that are expected to interfere with the cap to polyA interactions had no effect on the co-sedimentation pattern. Moreover, the 3′-UTR appeared to co-sediment with different regions from within the ORF. Taken together, these results indicate extensive physical associations between 3′-UTRs and their ORFs that vary between genes. This implies that polyribosomal mRNAs are in a compact configuration in vivo.     

The RNA helicase Ddx5/p68 binds to hUpf3 and enhances NMD of Ddx17/p72 and Smg5 mRNA

Non-sense-mediated mRNA decay (NMD) is a mechanism of translation-dependent mRNA surveillance in eukaryotes: it degrades mRNAs with premature termination codons (PTCs) and contributes to cellular homeostasis by downregulating a number of physiologically important mRNAs. In the NMD pathway, Upf proteins, a set of conserved factors of which Upf1 is the central regulator, recruit decay enzymes to promote RNA cleavage. In mammals, the degradation of PTC-containing mRNAs is triggered by the exon–junction complex (EJC) through binding of its constituents Upf2 and Upf3 to Upf1. The complex formed eventually induces translational repression and recruitment of decay enzymes. Mechanisms by which physiological mRNAs are targeted by the NMD machinery in the absence of an EJC have been described but still are discussed controversially. Here, we report that the DEAD box proteins Ddx5/p68 and its paralog Ddx17/p72 also bind the Upf complex by physical interaction with Upf3, thereby interfering with the binding of EJC. By activating the NMD machinery, Ddx5 is shown to regulate the expression of its own, Ddx17 and Smg5 mRNAs. For NMD triggering, the adenosine triphosphate-binding activity of Ddx5 and the 3′-untranslated region of substrate mRNAs are essential.     

Genome-Wide Identification of Alternative Splice Forms Down-Regulated by Nonsense-Mediated mRNA Decay in Drosophila

Alternative mRNA splicing adds a layer of regulation to the expression of thousands of genes in Drosophila melanogaster. Not all alternative splicing results in functional protein; it can also yield mRNA isoforms with premature stop codons that are degraded by the nonsense-mediated mRNA decay (NMD) pathway. This coupling of alternative splicing and NMD provides a mechanism for gene regulation that is highly conserved in mammals. NMD is also active in Drosophila, but its effect on the repertoire of alternative splice forms has been unknown, as has the mechanism by which it recognizes targets. Here, we have employed a custom splicing-sensitive microarray to globally measure the effect of alternative mRNA processing and NMD on Drosophila gene expression. We have developed a new algorithm to infer the expression change of each mRNA isoform of a gene based on the microarray measurements. This method is of general utility for interpreting splicing-sensitive microarrays and high-throughput sequence data. Using this approach, we have identified a high-confidence set of 45 genes where NMD has a differential effect on distinct alternative isoforms, including numerous RNA–binding and ribosomal proteins. Coupled alternative splicing and NMD decrease expression of these genes, which may in turn have a downstream effect on expression of other genes. The NMD–affected genes are enriched for roles in translation and mitosis, perhaps underlying the previously observed role of NMD factors in cell cycle progression. Our results have general implications for understanding the NMD mechanism in fly. Most notably, we found that the NMD–target mRNAs had significantly longer 3′ untranslated regions (UTRs) than the nontarget isoforms of the same genes, supporting a role for 3′ UTR length in the recognition of NMD targets in fly.     

m6A‐mediated alternative splicing coupled with nonsense‐mediated mRNA decay regulates SAM synthetase homeostasis

Alternative splicing of pre‐mRNAs can regulate gene expression levels by coupling with nonsense‐mediated mRNA decay (NMD). In order to elucidate a repertoire of mRNAs regulated by alternative splicing coupled with NMD (AS‐NMD) in an organism, we performed long‐read RNA sequencing of poly(A)⁺ RNAs from an NMD‐deficient mutant strain of Caenorhabditis elegans, and obtained full‐length sequences for mRNA isoforms from 259 high‐confidence AS‐NMD genes. Among them are the S‐adenosyl‐L‐methionine (SAM) synthetase (sams) genes sams‐3 and sams‐4. SAM synthetase activity autoregulates sams gene expression through AS‐NMD in a negative feedback loop. We furthermore find that METT‐10, the orthologue of human U6 snRNA methyltransferase METTL16, is required for the splicing regulation in␣vivo, and specifically methylates the invariant AG dinucleotide at the distal 3′ splice site (3′SS) in␣vitro. Direct RNA sequencing coupled with machine learning confirms m⁶A modification of endogenous sams mRNAs. Overall, these results indicate that homeostasis of SAM synthetase in C. elegans is maintained by alternative splicing regulation through m⁶A modification at the 3′SS of the sams genes.     

Knock-down of 25 kDa subunit of cleavage factor Im in Hela cells alters alternative polyadenylation within 3'-UTRs

Alternative polyadenylation leads to mRNAs with variable 3′ ends. Since a 3′-untranslated region (3′-UTR) often contains cis elements that impact stability or localization of mRNA or translation, selection of poly(A) sites in a 3′-UTR is regulated in mammalian cells. However, the molecular basis for alternative poly(A) site selection within a 3′-UTR has been unclear. Here we show involvement of cleavage factor Im (CFIm) in poly(A) site selection within a 3′-UTR. CFIm is a heterodimeric 3′ end-processing complex, which functions to assemble other processing factors on pre-mRNA in vitro. We knocked down 25 kDa subunit of CFIm (CFIm25) in HeLa cells and analyzed alternative poly(A) site selection of TIMP-2, syndecan2, ERCC6 and DHFR genes by northern blotting. We observed changes in the distribution of mRNAs in CFIm25 depleted cells, suggesting a role for CFIm in alternative poly(A) site selection. Furthermore, tissue specific analysis demonstrated that the CFIm25 gene gave rise to 1.1, 2.0 and 4.6 kb mRNAs. The 4.6 kb mRNA was ubiquitously expressed, while the 1.1 and 2.0 kb mRNAs were expressed in a tissue specific manner. We found three likely poly(A) sites in the CFIm25 3′-UTR, suggesting alternative polyadenylation. Our results indicate that alternative poly(A) site selection is a well-regulated process in vivo.     

The 3'-untranslated regions of cytoskeletal muscle mRNAs inhibit translation by activating the double-stranded RNA-dependent protein kinase PKR

Cytoskeletal proteins are associated with actin in the microfilaments and have a major role in microfilament assembly and function. The expression of some of these proteins has been implicated in cell growth and transformation. Specifically, the 3′-untranslated regions (3′-UTRs) of tropomyosin, troponin and cardiac actin can induce muscle cell differentiation and appear to function as tumor suppressors. These RNA sequences are predicted to fold to form secondary structures with extended stretches of duplex. We show that the 3′-UTRs of the cytoskeletal mRNAs interact with the RNA-binding domain of the RNA-activated protein kinase PKR. Correspondingly, these RNAs activate PKR in vitro and inhibit globin translation in the rabbit reticulocyte lysate translation system. These data are consistent with a mechanism whereby PKR mediates the differentiation- and tumor-related actions of the cytoskeletal 3′-UTR sequences.     

Estrogen-induced upregulation and 3′-UTR shortening of CDC6

3′-Untranslated region (UTR) shortening of mRNAs via alternative polyadenylation (APA) has important ramifications for gene expression. By using proximal APA sites and switching to shorter 3′-UTRs, proliferating cells avoid miRNA-mediated repression. Such APA and 3′-UTR shortening events may explain the basis of some of the proto-oncogene activation cases observed in cancer cells. In this study, we investigated whether 17 β-estradiol (E2), a potent proliferation signal, induces APA and 3′-UTR shortening to activate proto-oncogenes in estrogen receptor positive (ER+) breast cancers. Our initial probe based screen of independent expression arrays suggested upregulation and 3′-UTR shortening of an essential regulator of DNA replication, CDC6 (cell division cycle 6), upon E2 treatment. We further confirmed the E2- and ER-dependent upregulation and 3′UTR shortening of CDC6, which lead to increased CDC6 protein levels and higher BrdU incorporation. Consequently, miRNA binding predictions and dual luciferase assays suggested that 3′-UTR shortening of CDC6 was a mechanism to avoid 3′-UTR-dependent negative regulations. Hence, we demonstrated CDC6 APA induction by the proliferative effect of E2 in ER+ cells and provided new insights into the complex regulation of APA. E2-induced APA is likely to be an important but previously overlooked mechanism of E2-responsive gene expression.     

Regulation of ribonucleotide reductase M2 expression by the upstream AUGs

Ribonucleotide reductase catalyzes a rate-limiting reaction in DNA synthesis by converting ribonucleotides to deoxyribonucleotides. It consists of two subunits and the small one, M2 (or R2), plays an essential role in regulating the enzyme activity and its expression is finely controlled. Changes in the M2 level influence the dNTP pool and, thus, DNA synthesis and cell proliferation. M2 gene has two promoters which produce two major mRNAs with 5′-untranslated regions (5′-UTRs) of different lengths. In this study, we found that the M2 mRNAs with the short (63 nt) 5′-UTR can be translated with high efficiency whereas the mRNAs with the long (222 nt) one cannot. Examination of the long 5′-UTR revealed four upstream AUGs, which are in the same reading frame as the unique physiological translation initiation codon. Further analysis demonstrated that these upstream AUGs act as negative cis elements for initiation at the downstream translation initiation codon and their inhibitory effect on M2 translation is eIF4G dependent. Based on the findings of this study, we conclude that the expression of M2 is likely regulated by fine tuning the translation from the mRNA with a long 5′-UTR during viral infection and during the DNA replication phase of cell proliferation.