Daniel R. Schoenberg

Common SNP in pre-miR-146a decreases mature miR expression and predisposes to papillary thyroid carcinoma

Although papillary thyroid carcinoma (PTC) displays strong heritability, no predisposing germ-line mutations have been found. We show that a common G/C polymorphism (rs2910164) within the pre-miR-146a sequence reduced the amount of pre- and mature miR-146a from the C allele 1.9- and 1.8-fold, respectively, compared with the G allele. This is matched by a similar decrease in the amount of each pre-miR generated from the corresponding pri-miR-146a in an in vitro processing reaction. The C allele also interfered with the binding of a nuclear factor to pre-miR-146a. The reduction in miR-146a led to less efficient inhibition of target genes involved in the Toll-like receptor and cytokine signaling pathway (TRAF6, IRAK1), and PTC1 (also known as CCDC6 or H4), a gene frequently rearranged with RET proto-oncogene in PTC. In an association study of 608 PTC patients and 901 controls, we found marked differences in genotype distribution of rs2910164 (P = 0.000002), the GC heterozygous state being associated with an increased risk of acquiring PTC (odds ratio = 1.62, P = 0.000007), and both homozygous states protective with odds ratio = 0.42 for the CC genotype (P = 0.003) and odds ratio = 0.69 for the GG genotype (P = 0.0006). Moreover, 4.7% of tumors had undergone somatic mutations of the SNP sequence. Thus, our data suggest that a common polymorphism in pre-miR-146a affects the miR expression, contributes to the genetic predisposition to PTC, and plays a role in the tumorigenesis through somatic mutation. Preliminary evidence suggests that these effects are mediated through target genes whose expression is affected by the SNP status.

Regulation of cytoplasmic mRNA decay

Discoveries made over the past 20 years highlight the importance of mRNA decay as a means of modulating gene expression and thereby protein production. Up until recently, studies largely focused on identifying cis-acting sequences that serve as mRNA stability or instability elements, the proteins that bind these elements, how the process of translation influences mRNA decay and the ribonucleases that catalyse decay. Now, current studies have begun to elucidate how the decay process is regulated. This Review examines our current understanding of how mammalian cell mRNA decay is controlled by different signalling pathways and lays out a framework for future research.

A role for the eIF4E-binding protein 4E-T in P-body formation and mRNA decay

4E-transporter (4E-T) is one of several proteins that bind the mRNA 5′cap-binding protein, eukaryotic initiation factor 4E (eIF4E), through a conserved binding motif. We previously showed that 4E-T is a nucleocytoplasmic shuttling protein, which mediates the import of eIF4E into the nucleus. At steady state, 4E-T is predominantly cytoplasmic and is concentrated in bodies that conspicuously resemble the recently described processing bodies (P-bodies), which are believed to be sites of mRNA decay. In this paper, we demonstrate that 4E-T colocalizes with mRNA decapping factors in bona fide P-bodies. Moreover, 4E-T controls mRNA half-life, because its depletion from cells using short interfering RNA increases mRNA stability. The 4E-T binding partner, eIF4E, also is localized in P-bodies. 4E-T interaction with eIF4E represses translation, which is believed to be a prerequisite for targeting of mRNAs to P-bodies. Collectively, these data suggest that 4E-T interaction with eIF4E is a priming event in inducing messenger ribonucleoprotein rearrangement and transition from translation to decay.

Mycobacterium tuberculosis lipomannan blocks TNF biosynthesis by regulating macrophage MAPK-activated protein kinase 2 (MK2) and microRNA miR-125b

Contact of Mycobacterium tuberculosis (M.tb) with the immune system requires interactions between microbial surface molecules and host pattern recognition receptors. Major M.tb-exposed cell envelope molecules, such as lipomannan (LM), contain subtle structural variations that affect the nature of the immune response. Here we show that LM from virulent M.tb (TB-LM), but not from avirulent Myocobacterium smegmatis (SmegLM), is a potent inhibitor of TNF biosynthesis in human macrophages. This difference in response is not because of variation in Toll-like receptor 2-dependent activation of the signaling kinase MAPK p38. Rather, TB-LM stimulation leads to destabilization of TNF mRNA transcripts and subsequent failure to produce TNF protein. In contrast, SmegLM enhances MAPK-activated protein kinase 2 phosphorylation, which is critical for maintaining TNF mRNA stability in part by contributing microRNAs (miRNAs). In this context, human miRNA miR-125b binds to the 3′ UTR region of TNF mRNA and destabilizes the transcript, whereas miR-155 enhances TNF production by increasing TNF mRNA half-life and limiting expression of SHIP1, a negative regulator of the PI3K/Akt pathway. We show that macrophages incubated with TB-LM and live M.tb induce high miR-125b expression and low miR-155 expression with correspondingly low TNF production. In contrast, SmegLM and live M. smegmatis induce high miR-155 expression and low miR-125b expression with high TNF production. Thus, we identify a unique cellular mechanism underlying the ability of a major M.tb cell wall component, TB-LM, to block TNF biosynthesis in human macrophages, thereby allowing M.tb to subvert host immunity and potentially increase its virulence.

RNA helicase A is necessary for translation of selected messenger RNAs

RNA helicase A (RHA) is a highly conserved DEAD-box protein that activates transcription, modulates RNA splicing and binds the nuclear pore complex. The life cycle of typical mRNA involves RNA processing and translation after ribosome scanning of a relatively unstructured 5′ untranslated region (UTR). The precursor RNAs of retroviruses and selected cellular genes harbor a complex 5′ UTR and use a yet-to-be-identified host post-transcriptional effector to stimulate efficient translation. Here we show that RHA recognizes a structured 5′-terminal post-transcriptional control element (PCE) of a retrovirus and the JUND growth-control gene. RHA interacts with PCE RNA in the nucleus and cytoplasm, facilitates polyribosome association and is necessary for its efficient translation. Our results reveal a previously unidentified role for RHA in translation and implicate RHA as an integrative effector in the continuum of gene expression from transcription to translation.

Identification of a Cytoplasmic Complex That Adds a Cap onto 5'-Monophosphate RNA

ABSTRACT Endonuclease decay of nonsense-containing β-globin mRNA in erythroid cells generates 5′-truncated products that were reported previously to have a cap or caplike structure. We confirmed that this 5′ modification is indistinguishable from the cap on full-length mRNA, and Western blotting, immunoprecipitation, and active-site labeling identified a population of capping enzymes in the cytoplasm of erythroid and nonerythroid cells. Cytoplasmic capping enzyme sediments in a 140-kDa complex that contains a kinase which, together with capping enzyme, converts 5′-monophosphate RNA into 5′-GpppX RNA. Capping enzyme shows diffuse and punctate staining throughout the cytoplasm, and its staining does not overlap with P bodies or stress granules. Expression of inactive capping enzyme in a form that is restricted to the cytoplasm reduced the ability of cells to recover from oxidative stress, thus supporting a role for capping in the cytoplasm and suggesting that some mRNAs may be stored in an uncapped state.

Beta -Globin mRNA decay in erythroid cells: UG site-preferred endonucleolytic cleavage that is augmented by a premature termination codon.

Previous work showed that human β-globin mRNAs harboring a premature termination codon are degraded in the erythroid tissues of mice to products that lack sequences from the mRNA 5′ end but contain a 5′ cap-like structure. Whether these decay products are the consequence of endonucleolytic or 5′-to-3′ exonucleolytic activity is unclear. We report that this β-globin mRNA decay pathway is recapitulated in cultured mouse erythroleukemia (MEL) cells and targets nonsense-free mRNA to a lesser extent than nonsense-containing mRNA. S1 nuclease mapping and primer extension demonstrated that 70–80% of decay product 5′ ends contain a UG dinucleotide. Detection of upstream counterparts of these decay products indicates that they are generated by endonucleolytic activity. Both crude and partially purified polysome extracts prepared from MEL cells contain an endonucleolytic activity that generates decay products comparable to those observed in vivo. These data suggest that an endonuclease with preference for UG dinucleotides is involved in the degradation of nonsense-containing and, to a lesser extent, nonsense-free human β-globin mRNAs in mouse erythroid cells.

A polysomal ribonuclease involved in the destabilization of albumin mRNA is a novel member of the peroxidase gene family

We have purified an approximately 60 kDa endoribonuclease from Xenopus liver polysomes with properties expected for a messenger RNase involved in the estrogen-regulated destabilization of serum protein mRNAs (Dompenciel et al., 1995, J Biol Chem 270:6108-6118). The present report describes the cloning of this protein and its identification as a novel member of the peroxidase gene family. This novel enzyme, named polysomal RNase 1, or PMR-1 has 57% sequence identity with myeloperoxidase, and like that protein, appears to be processed from a larger precursor. Unlike myeloperoxidase, however, PMR-1 lacks N-linked oligosaccharide, heme, and peroxidase activity. Western blot and immunoprecipitation experiments using epitope-specific antibodies to the derived protein sequence confirm the identity of the cloned cDNA to the protein originally isolated from polysomes. The 80 kDa pre-PMR-1 expressed in a recombinant baculovirus was not processed to the 60 kDa form in Sf9 cells and lacks RNase activity. However, the baculovirus-expressed mature 60-kDa form of the enzyme has RNase activity. The recombinant protein is an endonuclease that shows selectivity for albumin versus ferritin mRNA. While it does not cleave at consensus APyrUGA elements, recombinant PMR-1 generates the same minor cleavage products from albumin mRNA as PMR-1 purified from liver. Finally, we show estrogen induces only a small increase in the amount of PMR-1. This result is consistent with earlier data suggesting estrogen activates mRNA decay through a posttranslational pathway.

Identification of a novel member of the pentraxin family in Xenopus laevis.

Pentraxins are a family of acute phase reactants. Two family members, C-reactive protein (CRP) and serum amyloid P component (SAP), are known in a range of mammalian species. CRP and SAP are both about 200 residues long, and arose from a gene duplication event, apparently before the divergence of the mammalian orders. To elucidate the origins of mammalian pentraxins, we have searched for pentraxin-coding genes in the amphibian Xenopus laevis. We have identified a gene determining a protein (XL-PXN1) which is about twice the size expected: the XL-PXN1 gene appears to be a fusion between regions encoding an amino-terminal peptide of unknown function and a carboxy-terminal pentraxin. The pentraxin domain is more divergent from CRP and SAP than they are from each other: it provides an outgroup for analysis of the evolution of mammalian pentraxins and confirms that putative CRP and SAP proteins partly characterized in non-vertebrate species cannot be true homologues of the mammalian proteins.

New ways of initiating translation in eukaryotes? [2](multiple letters)

This letter to the editor is a response by a large number of investigators in the field of protein synthesis to the minireview published by Dr. Kozak in Molecular and Cellular Biology (9). This minireview attempts to create significant doubts regarding the published literature that we believe are unwarranted and to bolster Dr. Kozak’s own point of view regarding translation initiation. We therefore take serious issue with the scholarliness of the Kozak minireview. As will be shown, the Kozak minireview contains numerous distortions of fact and of published data and selectively utilizes the published literature. In every field of research there are legitimate concerns regarding the interpretation of results and the reproducibility of certain published data. Several of the issues raised by Dr. Kozak are legitimate in this regard, but they are not new and have hardly gone unnoticed, having been raised in scholarly and critical reviews elsewhere. At issue here is not the right to critically question results and interpretations but rather whether the Kozak minireview is scholarly and its tone is professional. We point out that much of the work challenged in the Kozak minireview was published in Molecular and Cellular Biology, as well as other leading peer-reviewed journals, and forms a mainstream of research on protein synthesis which is taking place in scores of laboratories around the world. In this minireview, Dr. Kozak dismisses three novel mechanisms for translation initiation which have now been well studied and extensively documented. One mechanism is internal ribosome entry, which she rejects in favor of ribosome scanning, a mechanism for translation initiation which she proposed over 20 years ago. In ribosome scanning, it is proposed that the 40S small ribosome subunit enters the mRNA from the 5 cap and undergoes a linear 5 -to-3 search for the initiation codon, which is typically an AUG. Internal ribosome entry involves the internal association of ribosome subunits at or near the initiation codon without the need for entry from the 5 end of the mRNA. A second mechanism opposed by Dr. Kozak is the initiation of protein synthesis without Met-tRNA, a universal and key component, as shown for several insect virus mRNAs. The ability to carry out translation without this initiator tRNA, and from the A site of the ribosome, has enormous implications for our understanding of protein synthesis and its evolution. A third mechanism of translation initiation which Dr. Kozak takes issue with is known as ribosome shunting or discontinuous scanning, which combines features of 5 entry of ribosomes by scanning and the internal translocation of ribosome subunits without further scanning to the initiation codon. It is clear to a great many researchers, as represented by the signatory list below, that the initiation of protein synthesis in eukaryotes is dynamic and flexible, involving a variety of mechanisms that have evolved to meet the complex demands of eukaryotic cells and viruses. Dr. Kozak has spent more than 10 years in strenuous opposition to the evidence for viral internal ribosome entry and the recognition of specific viral cis-acting internal ribosome entry site (IRES) elements. The minireview now attempts to use almost entirely the same kinds of arguments against cellular IRESs and other means of nonscanning translation initiation that Dr. Kozak used previously in her unsuccessful efforts to disprove viral IRESs. Whether Dr. Kozak explicitly acknowledges internal ribosome entry as an established fact, at least for viruses, is not at all clear in the minireview, although she does compare translation functions to the encephalomyocarditis virus (EMCV) IRES, but without comment or acceptance. It would be fairer to the reader and more intellectually honest to explicitly acknowledge internal ribosome entry as an established fact, at least for viruses, or—if she still wishes to oppose the idea—to do so openly. Needless to say, it is now difficult to mount a convincing case for blanket repudiation of IRESs in the face of overwhelming data, including elegant and compelling evidence from viral IRES-dependent translation of a circular RNA (3), which was not cited in the Kozak review. It is not practical to document here all of the examples in which published results were inappropriately presented in the minireview by Dr. Kozak. We refer readers to a recent comprehensive review which summarizes the current evidence in support of viral and cellular IRES elements and alternate mechanisms of translation initiation in eukaryotes and briefly overviews some of the key techniques which were questioned by Dr. Kozak (7). Consequently, we list below just several specific examples which are emblematic of the serious issues which are of concern to us. Several reasons are described by Dr. Kozak for dismissing reports of cellular IRESs. Dr. Kozak argues that because cellular IRESs often represent modest translation increases over background levels, they result from fortuitous positioning of RNA sequences in experimental constructs. This argument ignores the evidence that IRESs have been shown to represent a range of activities from weak to strong and to function by a variety of mechanisms. Indeed, the expression of many cellular genes encoding regulatory proteins is often tightly controlled at multiple steps to guarantee that correct protein levels are achieved, which is not generally equivalent to high protein levels. An IRES may therefore be relatively weak, but in combination with other levels of gene control, it achieves significant or correct protein expression levels under different physiological conditions. One example is the IRES of the proto-oncogene c-sis, which encodes platelet-derived growth factor 2 (PDGF2). This IRES is activated severalfold during megakaryocyte differentiation, in conjunction with induction of PDGF2/c-sis gene expression during differentiation (1, 16). The modest translation stimulation directed by the cellular PDGF2 IRES fine tunes PDGF2/c-sis gene expression during differentiation. Similar mechanisms are likely employed by other critical regulatory genes and may have widespread implications for cellular growth and development. Thus, it is arbitrary to dismiss cellular IRES elements as physiologically irrelevant artifacts merely because their effects on translation are moderate. A more considered view is that regulatory elements act at all levels of gene control, including transcription, mRNA transport, and mRNA stability and translation, and permit exquisite control precisely because they involve multiple and modest additive effects which can be independently combined and regulated. There are several functional ways to study IRESs. Construction of a dicistronic mRNA containing an internal downstream second open reading frame that is ordinarily not translated is typically used to detect IRES activity. Other approaches include insertion of very stable, translation-blocking hairpin structures upstream of the IRES and biochemical detection of IRES interaction with initiation factors and ribosome subunits. Important control studies must be performed to validate the integrity of the dicistronic mRNA and to exclude the presence of cryptic promoters or aberrant splicing that could lead to