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.     

C. elegans and H. sapiens mRNAs with edited 3' UTRs are present on polysomes

Adenosine deaminases that act on RNA (ADARs) are editing enzymes that convert adenosine to inosine in double-stranded RNA (dsRNA). ADARs sometimes target codons so that a single mRNA yields multiple protein isoforms. However, ADARs most often target noncoding regions of mRNAs, such as untranslated regions (UTRs). To understand the function of extensive double-stranded 3′ UTR structures, and the inosines within them, we monitored the fate of reporter and endogenous mRNAs that include structured 3′ UTRs in wild-type Caenorhabditis elegans and in strains with mutations in the ADAR genes. In general, we saw little effect of editing on stability or translatability of mRNA, although in one case an ADR-1 dependent effect was observed. Importantly, whereas previous studies indicate that inosine-containing RNAs are retained in the nucleus, we show that both C. elegans and Homo sapiens mRNAs with edited, structured 3′ UTRs are present on translating ribosomes.     

DAZL Relieves miRNA-Mediated Repression of Germline mRNAs by Controlling Poly(A) Tail Length in Zebrafish

Background

During zebrafish embryogenesis, microRNA (miRNA) miR-430 contributes to restrict Nanos1 and TDRD7 to primordial germ cells (PGCs) by inducing mRNA deadenylation, mRNA degradation, and translational repression of nanos1 and tdrd7 mRNAs in somatic cells. The nanos1 and tdrd7 3′UTRs include cis-acting elements that allow activity in PGCs even in the presence of miRNA-mediated repression.

Methodology/Principal Findings

Using a GFP reporter mRNA that was fused with tdrd7 3′UTR, we show that a germline-specific RNA-binding protein DAZ-like (DAZL) can relieve the miR-430-mediated repression of tdrd7 mRNA by inducing poly(A) tail elongation (polyadenylation) in zebrafish. We also show that DAZL enhances protein synthesis via the 3′UTR of dazl mRNA, another germline mRNA targeted by miR-430.

Conclusions/Significance

Our present study indicated that DAZL acts as an “anti-miRNA factor” during vertebrate germ cell development. Our data also suggested that miRNA-mediated regulation can be modulated on specific target mRNAs through the poly(A) tail control.     

Messenger RNAs located in myelin sheath assembly sites

The targeting of mRNAs to specific subcellular locations is believed to facilitate the rapid and selective incorporation of their protein products into complexes that may include membrane organelles. In oligodendrocytes, mRNAs that encode myelin basic protein (MBP) and select myelin-associated oligodendrocytic basic proteins (MOBPs) locate in myelin sheath assembly sites (MSAS). To identify additional mRNAs located in MSAS, we used a combination of subcellular fractionation and suppression subtractive hybridization. More than 50% of the 1,080 cDNAs that were analyzed were derived from MBP or MOBP mRNAs, confirming that the method selected mRNAs enriched in MSAS. Of 90 other cDNAs identified, most represent one or more mRNAs enriched in rat brain myelin. Five cDNAs, which encode known proteins, were characterized for mRNA size(s), enrichment in myelin, and tissue and developmental expression patterns. Two of these, peptidylarginine deiminase and ferritin heavy chain, have recognized roles in myelination. The corresponding mRNAs were of different sizes than the previously identified mRNA, and they had tissue and development expression patterns that were indistinguishable from those of MBP mRNA. Three other cDNAs recognize mRNAs whose proteins (SH3p13, KIF1A, and dynein light intermediate chain) are involved in membrane biogenesis. Although enriched in myelin, the tissue and developmental distribution patterns of these mRNAs differed from those of MBP mRNA. Six other cDNAs, which did not share significant sequence homology to known mRNAs, were also examined. The corresponding mRNAs were highly enriched in myelin, and four had tissue and developmental distribution patterns indistinguishable from those of MBP mRNA. These studies demonstrate that MSAS contain a diverse population of mRNAs, whose locally synthesized proteins are placed to contribute to myelin sheath assembly and maintenance. Characterization of these mRNAs and proteins will help provide a comprehensive picture of myelin sheath assembly.     

A Family of Insulin-Like Growth Factor II mRNA-Binding Proteins Represses Translation in Late Development

Insulin-like growth factor II (IGF-II) is a major fetal growth factor. The IGF-II gene generates multiple mRNAs with different 5′ untranslated regions (5′ UTRs) that are translated in a differential manner during development. We have identified a human family of three IGF-II mRNA-binding proteins (IMPs) that exhibit multiple attachments to the 5′ UTR from the translationally regulated IGF-II leader 3 mRNA but are unable to bind to the 5′ UTR from the constitutively translated IGF-II leader 4 mRNA. IMPs contain the unique combination of two RNA recognition motifs and four hnRNP K homology domains and are homologous to the Xenopus Vera and chicken zipcode-binding proteins. IMP localizes to subcytoplasmic domains in a growth-dependent and cell-specific manner and causes a dose-dependent translational repression of IGF-II leader 3 –luciferase mRNA. Mouse IMPs are produced in a burst at embryonic day 12.5 followed by a decline towards birth, and, similar to IGF-II, IMPs are especially expressed in developing epithelia, muscle, and placenta in both mouse and human embryos. The results imply that cytoplasmic 5′ UTR-binding proteins control IGF-II biosynthesis during late mammalian development.     

An evolutionary conserved pattern of 18S rRNA sequence complementarity to mRNA 5′ UTRs and its implications for eukaryotic gene translation regulation

There are several key mechanisms regulating eukaryotic gene expression at the level of protein synthesis. Interestingly, the least explored mechanisms of translational control are those that involve the translating ribosome per se, mediated for example via predicted interactions between the ribosomal RNAs (rRNAs) and mRNAs. Here, we took advantage of robustly growing large-scale data sets of mRNA sequences for numerous organisms, solved ribosomal structures and computational power to computationally explore the mRNA–rRNA complementarity that is statistically significant across the species. Our predictions reveal highly specific sequence complementarity of 18S rRNA sequences with mRNA 5′ untranslated regions (UTRs) forming a well-defined 3D pattern on the rRNA sequence of the 40S subunit. Broader evolutionary conservation of this pattern may imply that 5′ UTRs of eukaryotic mRNAs, which have already emerged from the mRNA-binding channel, may contact several complementary spots on 18S rRNA situated near the exit of the mRNA binding channel and on the middle-to-lower body of the solvent-exposed 40S ribosome including its left foot. We discuss physiological significance of this structurally conserved pattern and, in the context of previously published experimental results, propose that it modulates scanning of the 40S subunit through 5′ UTRs of mRNAs.     

Activation of cap-independent translation by variant eukaryotic initiation factor 4G in vivo

Protein synthesis is tightly controlled by assembly of an intricate ribonucleoprotein complex at the m⁷GTP-cap on eukaryotic mRNAs. Ensuing linear scanning of the 5′ untranslated region (UTR) is believed to transfer the preinitiation complex to the initiation codon. Eukaryotic mRNAs are characterized by significant 5′ UTR heterogeneity, raising the possibility of differential control of translation initiation rate at individual mRNAs. Curiously, many mRNAs with unconventional, highly structured 5′ UTRs encode proteins with central biological roles in growth control, metabolism, or stress response. The 5′ UTRs of such mRNAs may influence protein synthesis rate in multiple ways, but most significantly they have been implicated in mediating alternative means of translation initiation. Cap-independent initiation bypasses strict control over the formation of initiation intermediates at the m⁷GTP cap. However, the molecular mechanisms that favor alternative means of ribosome recruitment are not understood. Here we provide evidence that eukaryotic initiation factor (eIF) 4G controls cap-independent translation initiation at the c-myc and vascular endothelial growth factor (VEGF) 5′ UTRs in vivo. Cap-independent translation was investigated in tetracycline-inducible cell lines expressing either full-length eIF4G or a C-terminal fragment (Ct) lacking interaction with eIF4E and poly(A) binding protein. Expression of Ct, but not intact eIF4G, potently stimulated cap-independent initiation at the c-myc/VEGF 5′ UTRs. In vitro RNA-binding assays suggest that stimulation of cap-independent translation initiation by Ct is due to direct association with the c-myc/VEGF 5′ UTR, enabling 43S preinitiation complex recruitment. Our work demonstrates that variant translation initiation factors enable unconventional translation initiation at mRNA subsets with distinct structural features.     

MS2 coat proteins bound to yeast mRNAs block 5′ to 3′ degradation and trap mRNA decay products: implications for the localization of mRNAs by MS2-MCP system

Arrays of MS2 binding sites are placed into mRNAs and are commonly used to visualize the localization of mRNAs in vivo by the expression of an MS2-GFP fusion protein. In Saccharomyces cerevisiae, we observed that arrays of MS2 binding sites inhibit 5′ to 3′ degradation of the mRNA in yeast cells and lead to the accumulation of a 3′ mRNA fragment containing the MS2 binding sites. This accumulation can be dependent on the binding of the MS2 stem loops (MS2-SL) by MS2 coat proteins (MCPs). Since such decay fragments can still bind MCP-GFP, the localization of such mRNA fragments can complicate the interpretation of the localization of full-length mRNA in vivo.     

Myelin-Associated Oligodendrocytic Basic Protein mRNAs Reside at Different Subcellular Locations

The mRNAs for two myelin proteins, myelin basic protein (MBP) and myelin-associated oligodendrocytic basic protein (MOBP)-81A, are uniquely located at sites where myelin sheaths are assembled. Here, we use subcellular fractionation to show that four MOBP mRNAs, like MBP mRNA, are located at sites of myelin sheath assembly, and that three other MOBP mRNAs are located in oligodendrocyte soma. The MOBP-81 protein is found in myelin and in another subcellular fraction, whereas other myelin proteins, including MBP, 2',3'-cyclic nucleotide 3'-phosphodiesterase, and myelin-associated glycoprotein, are largely restricted to myelin. Different MBP mRNAs are generated by alternative splicing. All of them contain an RNA transport sequence (RTS) that directs them to sites in oligodendrocytes, where myelin sheaths are assembled. Consequently, all are enriched in myelin. After fractionation, four MOBP mRNAs, MOBP-71, MOBP-81A, MOBP-99, and MOBP-169 (identified in this study), are enriched in myelin. These mRNAs contain a common exon, exon 8b, which has a nucleotide sequence that is similar to MBP mRNA RTS. This sequence likely directs these mRNAs to sites of myelin sheath assembly. Three other MOBP mRNAs, MOBP-69, MOBP-81B, and MOBP-170, lack this exon. Their subcellular distribution indicates that they are largely retained in oligodendrocyte soma. We conclude that the distribution of MOBPs in oligodendrocytes is strongly influenced by alternative splicing of the corresponding mRNAs.     

Lamin-binding Fragment of LAP2 Inhibits Increase in Nuclear Volume during the Cell Cycle and Progression into S Phase

Myelin basic protein (MBP) mRNA is localized to myelin produced by oligodendrocytes of the central nervous system. MBP mRNA microinjected into oligodendrocytes in primary culture is assembled into granules in the perikaryon, transported along the processes, and localized to the myelin compartment. In this work, microinjection of various deleted and chimeric RNAs was used to delineate regions in MBP mRNA that are required for transport and localization in oligodendrocytes. The results indicate that transport requires a 21-nucleotide sequence, termed the RNA transport signal (RTS), in the 3′ UTR of MBP mRNA. Homologous sequences are present in several other localized mRNAs, suggesting that the RTS represents a general transport signal in a variety of different cell types. Insertion of the RTS from MBP mRNA into nontransported mRNAs, causes the RNA to be transported to the oligodendrocyte processes. Localization of mRNA to the myelin compartment requires an additional element, termed the RNA localization region (RLR), contained between nucleotide 1,130 and 1,473 in the 3′ UTR of MBP mRNA. Computer analysis predicts that this region contains a stable secondary structure. If the coding region of the mRNA is deleted, the RLR is no longer required for localization, and the region between nucleotide 667 and 953, containing the RTS, is sufficient for both RNA transport and localization. Thus, localization of coding RNA is RLR dependent, and localization of noncoding RNA is RLR independent, suggesting that they are localized by different pathways.