The Mytilus chilensis Steamer-like Element-1 Retrotransposon Antisense mRNA Harbors an Internal Ribosome Entry Site That Is Modulated by hnRNPK.

LTR-retrotransposons are transposable elements characterized by the presence of long terminal repeats (LTRs) directly flanking an internal coding region. They share genome organization and replication strategies with retroviruses. Steamer-like Element-1 (MchSLE-1) is an LTR-retrotransposon identified in the genome of the Chilean blue mussel Mytilus chilensis. MchSLE-1 is transcribed; however, whether its RNA is also translated and the mechanism underlying such translation remain to be elucidated. Here, we characterize the MchSLE-1 translation mechanism. We found that the MchSLE-1 5' and 3'LTRs command transcription of sense and antisense RNAs, respectively. Using luciferase reporters commanded by the untranslated regions (UTRs) of MchSLE-1, we found that in vitro 5'UTR sense is unable to initiate translation, whereas the antisense 5'UTR initiates translation even when the eIF4E-eIF4G interaction was disrupted, suggesting the presence of an internal ribosomal entry site (IRES). The antisense 5'UTR IRES activity was tested using bicistronic reporters. The antisense 5'UTR has IRES activity only when the mRNA is transcribed in the nucleus, suggesting that nuclear RNA-binding proteins are required to modulate its activity. Indeed, heterogeneous nuclear ribonucleoprotein K (hnRNPK) was identified as an IRES trans-acting factor (ITAF) of the MchSLE-1 IRES. To our knowledge, this is the first report describing an IRES in an antisense mRNA derived from a mussel LTR-retrotransposon.

Heterogeneous nuclear ribonucleoprotein K promotes cap-independent translation initiation of retroviral mRNAs.

Translation initiation of the human immunodeficiency virus-type 1 (HIV-1) genomic mRNA (vRNA) is cap-dependent or mediated by an internal ribosome entry site (IRES). The HIV-1 IRES requires IRES-transacting factors (ITAFs) for function. In this study, we evaluated the role of the heterogeneous nuclear ribonucleoprotein K (hnRNPK) as a potential ITAF for the HIV-1 IRES. In HIV-1-expressing cells, the depletion of hnRNPK reduced HIV-1 vRNA translation. Furthermore, both the depletion and overexpression of hnRNPK modulated HIV-1 IRES activity. Phosphorylations and protein arginine methyltransferase 1 (PRMT1)-induced asymmetrical dimethylation (aDMA) of hnRNPK strongly impacted the protein's ability to promote the activity of the HIV-1 IRES. We also show that hnRNPK acts as an ITAF for the human T cell lymphotropic virus-type 1 (HTLV-1) IRES, present in the 5'UTR of the viral sense mRNA, but not for the IRES present in the antisense spliced transcript encoding the HTLV-1 basic leucine zipper protein (sHBZ). This study provides evidence for a novel role of the host hnRNPK as an ITAF that stimulates IRES-mediated translation initiation for the retroviruses HIV-1 and HTLV-1.

Alternative splicing of its 5'-UTR limits CD20 mRNA translation and enables resistance to CD20-directed immunotherapies.

Aberrant skipping of coding exons in CD19 and CD22 compromises the response to immunotherapy in B-cell malignancies. Here, we showed that the MS4A1 gene encoding human CD20 also produces several messenger RNA (mRNA) isoforms with distinct 5' untranslated regions. Four variants (V1-4) were detected using RNA sequencing (RNA-seq) at distinct stages of normal B-cell differentiation and B-lymphoid malignancies, with V1 and V3 being the most abundant. During B-cell activation and Epstein-Barr virus infection, redirection of splicing from V1 to V3 coincided with increased CD20 positivity. Similarly, in diffuse large B-cell lymphoma, only V3, but not V1, correlated with CD20 protein levels, suggesting that V1 might be translation-deficient. Indeed, the longer V1 isoform contained upstream open reading frames and a stem-loop structure, which cooperatively inhibited polysome recruitment. By modulating CD20 isoforms with splice-switching morpholino oligomers, we enhanced CD20 expression and anti-CD20 antibody rituximab-mediated cytotoxicity in a panel of B-cell lines. Furthermore, reconstitution of CD20-knockout cells with V3 mRNA led to the recovery of CD20 positivity, whereas V1-reconstituted cells had undetectable levels of CD20 protein. Surprisingly, in vitro CD20-directed chimeric antigen receptor T cells were able to kill both V3- and V1-expressing cells, but the bispecific T-cell engager mosunetuzumab was only effective against V3-expressing cells. To determine whether CD20 splicing is involved in immunotherapy resistance, we performed RNA-seq on 4 postmosunetuzumab follicular lymphoma relapses and discovered that in 2 of them, the downregulation of CD20 was accompanied by a V3-to-V1 shift. Thus, splicing-mediated mechanisms of epitope loss extend to CD20-directed immunotherapies.

First report of bovine viral diarrhea virus subgenotypes 1d and 1e in southern Chile.

Bovine viral diarrhea virus (BVDV) affects cattle worldwide causing severe productive and economic loss. In this study, we investigated the subgenotypes of BVDV circulating in cattle samples from the Aysén region, an active cattle breeding area located in southern Chile. Partial amplification of the 5' untranslated region (UTR) was performed by polymerase chain reaction (PCR), and twelve samples were analyzed by Sanger sequencing and phylogenetic analysis. Eight samples were identified as belonging to Pestivirus bovis subgenotype 1e, three to 1-b, and one to 1-d. The phylogenetic analyses performed revealed a marked distance between these now-identified strains and those previously reported in the country. These findings support the need to continually expand the analysis of the variability of the viral phylogeny for the currently circulating BVDV strains and to update the vaccines recommended for this livestock area and surrounding areas.

Novel genomic targets for proper subtyping of bovine viral diarrhea virus 1 (BVDV-1) and BVDV-2.

Whole-genome phylogenetic analysis, the most suitable strategy for subtyping bovine viral diarrhea virus 1 (BVDV-1) and BVDV-2, is not feasible for many laboratories. Consequently, BVDV isolates/strains have been frequently subtyped based on analysis of single genomic regions, mainly the 5' untranslated region (UTR). This approach, however, may lead to inaccurate and/or poorly statistically supported viral classification. Herein, we describe novel primer sets whose amplicons may be easily sequenced and used for BVDV subtyping. Initially, genomic regions previously described as the most suitable targets for BVDV subtyping were analyzed for design of high-coverage primers. The putative amplicons were analyzed in silico for their suitability to reproduce the phylogenetic classification of 118 BVDV-1 and 88 BVDV-2 complete/near-complete genomes (CNCGs) (GenBank). This analysis was also performed considering the region amplifiable by primers HCV90-368, 324-326 and BP189-389 (5'UTR), which have been used for BVDV diagnosis and/or classification. After confirming the agreement between the analyses of our primers' amplicon versus the CNCGs, we optimized the RT-PCRs and evaluated their performance for amplification of BVDV isolates/strains (n = 35 for BVDV-1; n = 33 for BVDV-2). Among the potential targets for BVDV subtyping, we designed high-coverage primers for NS3-NS4A (BVDV-1) (526 bp amplicon) and NS5B (BVDV-2) (728 bp). The classification based on these regions fully reproduced the subtyping of all CNCGs. On the other hand, subtyping based on the putative amplicons from primers HCV90-368, 324-326 and BP189-389 showed disagreements in relation the CNCG analysis. The NS3-NS4A and NS5B primers also allowed the amplification of all BVDV isolates/strains tested. Finally, we suggest the use of these primers in future phylogenetic and epidemiological studies of BVDVs.

5'/3' RACE method for sequencing the 5' and 3' untranslated regions of Zika virus.

The spread of Zika virus (ZIKV) from the African continent to the Americas promoted its molecular evolution, as reflected by mutations in its RNA genome. Most of the ZIKV genome sequences in the GenBank database have incomplete 5' and 3' UTR sequences, reflecting the deficiency of whole-genome sequencing technologies to resolve the sequences of the genome ends. We modified a protocol for rapid amplification of cDNA ends (RACE) to determine the complete sequences of the 5' and 3' UTRs of a previously reported ZIKV isolate (GenBank no. MH544701.1). This strategy is useful for determining 5' and 3' UTR sequences of ZIKV isolates and will be useful for comparative genomics applications.

Trans-splicing in the cestode Hymenolepis microstoma is constitutive across the life cycle and depends on gene structure and composition.

Spliced leader (SL) trans-splicing is a key process during mRNA maturation of many eukaryotes, in which a short sequence (SL) is transferred from a precursor SL-RNA into the 5' region of an immature mRNA. This mechanism is present in flatworms, in which it is known to participate in the resolution of polycistronic transcripts. However, most trans-spliced transcripts are not part of operons, and it is not clear if this process may participate in additional regulatory mechanisms in this group. In this work, we present a comprehensive analysis of SL trans-splicing in the model cestode Hymenolepis microstoma. We identified four different SL-RNAs which are indiscriminately trans-spliced to 622 gene models. SL trans-splicing is enriched in constitutively expressed genes and does not appear to be regulated throughout the life cycle. Operons represented at least 20% of all detected trans-spliced gene models, showed conservation to those of the cestode Echinococcus multilocularis, and included complex loci such as an alternative operon (processed as either a single gene through cis-splicing or as two genes of a polycistron). Most insertion sites were identified in the 5' untranslated region (UTR) of monocistronic genes. These genes frequently contained introns in the 5' UTR, in which trans-splicing used the same acceptor sites as cis-splicing. These results suggest that, unlike other eukaryotes, trans-splicing is associated with internal intronic promoters in the 5' UTR, resulting in transcripts with strong splicing acceptor sites without competing cis-donor sites, pointing towards a simple mechanism driving the evolution of novel SL insertion sites.

YELLOW ALERT: Persistent Yellow Fever Virus Circulation among Non-Human Primates in Urban Areas of Minas Gerais State, Brazil (2021-2023).

Yellow fever virus (YFV) is the agent of yellow fever (YF), which affects both humans and non-human primates (NHP). Neotropical NHP are highly susceptible to YFV and considered sentinels for YFV circulation. Brazil faced a significant YF outbreak in 2017-2018, with over 2000 human cases and 2000 epizootics cases, mainly in the State of Minas Gerais, Brazil. This study aimed to investigate whether YFV circulation persisted in NHP after the human outbreak had subsided. To this end, NHP carcass samples collected in Minas Gerais from 2021 to 2023 were screened for YFV. RNA was extracted from tissue fragments and used in RT-qPCR targeting the YFV 5'UTR. Liver and lung samples from 166 animals were tested, and the detection of the ß-actin mRNA was used to ensure adequacy of RNA isolation. YFV RNA was detected in the liver of 18 NHP carcasses collected mainly from urban areas in 2021 and 2022. YFV positive NHP were mostly represented by Callithrix, from 5 out of the 12 grouped municipalities (mesoregions) in Minas Gerais state. These findings reveal the continued YFV circulation in NHP in urban areas of Minas Gerais during 2021 and 2022, with the attendant risk of re-establishing the urban YFV cycle.

Genetic and phenotypic stability of poliovirus shed from infants who received novel type 2 or Sabin type 2 oral poliovirus vaccines in Panama: an analysis of two clinical trials.

BACKGROUND: Sabin strains used in oral poliovirus vaccines (OPV) can revert to virulence and, in rare instances, cause disease or generate vaccine-derived strains leading to outbreaks in areas of low immunisation coverage. A novel OPV2 (nOPV2) was designed to stabilise the viral genome against reversion and reduce recombination events that might lead to virulent strains. In this study, we evaluated the genetic and phenotypic stability of shed poliovirus following administration of one dose of monovalent OPV2 (mOPV2) or nOPV2 to infants aged 18-22 weeks. METHODS: In two similarly designed clinical trials (NCT02521974 and NCT03554798) conducted in Panama, infants aged 18-22-weeks, after immunisation with three doses of bivalent OPV (types 1 and 3) and one dose of inactivated poliovirus vaccine, were administered one or two doses of mOPV2 or nOPV2. In this analysis of two clinical trials, faecally shed polioviruses following one dose of mOPV2 or nOPV2 were isolated from stools meeting predetermined criteria related to sample timing and viral presence and quantity and assessed for nucleotide polymorphisms using next-generation sequencing. A transgenic mouse neurovirulence test was adapted to assess the effect of the possible phenotypic reversion of shed mOPV2 and nOPV2 with a logistic regression model. FINDINGS: Of the 91 eligible samples, 86 were able to be sequenced, with 72 evaluated in the transgenic mouse assay. Sabin-2 poliovirus reverts rapidly at nucleotide 481, the primary attenuation site in domain V of the 5' untranslated region of the genome. There was no evidence of neurovirulence-increasing polymorphisms in domain V of shed nOPV2. Reversion of shed Sabin-2 virus corresponded with unadjusted paralysis rates of 47·6% at the 4 log10 50% cell culture infectious dose (CCID50) and 76·7% at the 5 log10 CCID50 inoculum levels, with rates of 2·8% for 4 log10 CCID50 and 11·8% for 5 log10 CCID50 observed for shed nOPV2 samples. The estimated adjusted odds ratio at 4·5 log10 of 0·007 (95% CI 0·002-0·023; p<0·0001) indicates significantly reduced odds of mouse paralysis from virus obtained from nOPV2 recipients compared with mOPV2 recipients. INTERPRETATION: The data indicate increased genetic stability of domain V of nOPV2 relative to mOPV2, with significantly lower neurovirulence of shed nOPV2 virus compared with shed mOPV2. While this vaccine is currently being deployed under an emergency use listing, the data on the genetic stability of nOPV2 will support further regulatory and policy decision-making regarding use of nOPV2 in outbreak responses. FUNDING: Bill & Melinda Gates Foundation.

Brazilian family with hyperferritinemia-cataract syndrome: case report.

Hereditary hyperferritinemia-cataract syndrome is a rare autosomal dominant disease caused by a genetic mutation in the iron responsive element in the 5' untranslated region of the ferritin light chain gene. Hereditary hyperferritinemia-cataract syndrome is characterized by elevated serum ferritin levels and bilateral cataract development early in life and may be misdiagnosed as hemochromatosis. This case report describes a Brazilian family with a clinical diagnosis of hereditary hyperferritinemia-cataract syndrome, which was submitted to ferritin light chain gene sequencing. The genetic mutation c.-164C>G was identified in the 5' untranslated region. In conclusion, genetic testing can be used for accurate diagnosis of hereditary hyperferritinemia-cataract syndrome to avoid misdiagnosis of hemochromatosis, other diseases associated with iron overload or ophthalmic diseases.

A new (old) bovine viral diarrhea virus 2 subtype: BVDV-2e.

Bovine pestiviruses are members of the species Pestivirus A (bovine viral diarrhea virus 1, BVDV-1), Pestivirus B (BVDV-2) or Pestivirus H (HoBiPeV). To date, BVDV-2 isolates/strains have been classified into three subtypes (a-c) by phylogenetic analysis, and an additional subtype (d) has been proposed based on 5' untranslated region (UTR) secondary structures. In a previous study, we identified some BVDV-2 sequences in the GenBank database that could not be classified as subtype a, b or c by phylogenetic analysis of their genomes, UTRs or individual genes. Here, we performed a detailed study of these sequences and assessed whether they might represent a distinct BVDV-2 subtype. Initially, we collected 85 BVDV-2 complete/near-complete genomes (CNCGs) from GenBank and performed a "proof of equivalence" between phylogenetic analyses based on CNCGs and open reading frames (ORFs), which showed that ORFs may be reliably used as a reference target for BVDV-2 phylogeny, allowing us to increase our dataset to 139 sequences. Among these, we found seven sequences that could not be classified as BVDV-2a-c. The same was observed in the phylogenetic analysis of CNCGs and viral genes. In addition, the seven non-BVDV-2a-c sequences formed a distinct cluster in all phylogenetic trees, which we propose to term BVDV-2e. BVDV-2e also showed 44 amino acid changes compared to BVDV-2a-c, 20 of which are in well-defined positions. Importantly, an additional phylogenetic analysis including BVDV-2d and a pairwise comparison of BVDV-2e and BVDV-2d sequences also supported the difference between these subtypes. Finally, we propose the recognition of BVDV-2e as a distinct BVDV-2 subtype and encourage its inclusion in future phylogenetic analyses to understand its distribution and evolution.

Expression and characterisation of Fmr1 splice variants during folliculogenesis in the rat.

CONTEXT: The FMR1 gene consists of 17 exons and codes for the FMRP protein. FMR1 is involved in four genetic disorders depending on the CGG repeats length in its 5'UTR: the full mutation is responsible for the Fragile X syndrome while the premutation is associated with the Fragile X-associated Tremor/Ataxia Syndrome, Fragile X-associated Primary Ovarian Insufficiency (FXPOI) and Fragile X-associated neuropsychiatric disorders. FMR1 presents multiple isoforms resulting from skipping of exons 12 and 14 and the use of alternative splice sites in exons 15 and 17. AIMS: To investigate the expression of Fmr1 splicing variants during folliculogenesis in the rat. METHODS: We used preantral, early antral and preovulatory follicles to isolate RNA and characterise, by fluorescent PCR followed by sequencing, all the isoforms present in the different follicular stages. KEY RESULTS: We identified two isoforms resulting from splicing of exon 12, six isoforms resulting from splicing of exon 14 and 15 and one isoform for exon 17. CONCLUSIONS: The expression levels of the isoforms vary within each follicular stage but not between different stages of folliculogenesis. Importantly, we identify for the first time in rat, an isoform that contains exon 12 and two isoforms, one that includes and one that excludes exon 14 and use the third acceptor site in exon 15. IMPLICATIONS: Characterisation of the different FMR1 variants expressed during folliculogenesis will help to understand the potential distinct cellular roles of each of them and the possible implication in the development of FXPOI.

Role of MicroRNA-7 (MiR-7) in Cancer Physiopathology.

miRNAs are non-coding RNA sequences of approximately 22 nucleotides that interact with genes by inhibiting their translation through binding to their 3' or 5' UTR regions. Following their discovery, the role they play in the development of various pathologies, particularly cancer, has been studied. In this context, miR-7 is described as an important factor in the development of cancer because of its role as a tumor suppressor, regulating a large number of genes involved in the development and progression of cancer. Recent data support the function of miR-7 as a prognostic biomarker in cancer, and miR-7 has been proposed as a strategy in cancer therapy. In this work, the role of miR-7 in various types of cancer is reviewed, illustrating its regulation, direct targets, and effects, as well as its possible relationship to the clinical outcome of cancer patients.

Epitranscriptomic regulation of HIV-1 full-length RNA packaging.

During retroviral replication, the full-length RNA serves both as mRNA and genomic RNA. However, the mechanisms by which the HIV-1 Gag protein selects the two RNA molecules that will be packaged into nascent virions remain poorly understood. Here, we demonstrate that deposition of N6-methyladenosine (m6A) regulates full-length RNA packaging. While m6A deposition by METTL3/METTL14 onto the full-length RNA was associated with increased Gag synthesis and reduced packaging, FTO-mediated demethylation promoted the incorporation of the full-length RNA into viral particles. Interestingly, HIV-1 Gag associates with the RNA demethylase FTO in the nucleus and contributes to full-length RNA demethylation. We further identified two highly conserved adenosines within the 5'-UTR that have a crucial functional role in m6A methylation and packaging of the full-length RNA. Together, our data propose a novel epitranscriptomic mechanism allowing the selection of the HIV-1 full-length RNA molecules that will be used as viral genomes.

Polypyrimidine-Tract-Binding Protein Isoforms Differentially Regulate the Hepatitis C Virus Internal Ribosome Entry Site.

Translation initiation of the hepatitis C virus (HCV) mRNA depends on an internal ribosome entry site (IRES) that encompasses most of the 5'UTR and includes nucleotides of the core coding region. This study shows that the polypyrimidine-tract-binding protein (PTB), an RNA-binding protein with four RNA recognition motifs (RRMs), binds to the HCV 5'UTR, stimulating its IRES activity. There are three isoforms of PTB: PTB1, PTB2, and PTB4. Our results show that PTB1 and PTB4, but not PTB2, stimulate HCV IRES activity in HuH-7 and HEK293T cells. In HuH-7 cells, PTB1 promotes HCV IRES-mediated initiation more strongly than PTB4. Mutations in PTB1, PTB4, RRM1/RRM2, or RRM3/RRM4, which disrupt the RRM's ability to bind RNA, abrogated the protein's capacity to stimulate HCV IRES activity in HuH-7 cells. In HEK293T cells, PTB1 and PTB4 stimulate HCV IRES activity to similar levels. In HEK293T cells, mutations in RRM1/RRM2 did not impact PTB1's ability to promote HCV IRES activity; and mutations in PTB1 RRM3/RRM4 domains reduced, but did not abolish, the protein's capacity to stimulate HCV IRES activity. In HEK293T cells, mutations in PTB4 RRM1/RRM2 abrogated the protein's ability to promote HCV IRES activity, and mutations in RRM3/RRM4 have no impact on PTB4 ability to enhance HCV IRES activity. Therefore, PTB1 and PTB4 differentially stimulate the IRES activity in a cell type-specific manner. We conclude that PTB1 and PTB4, but not PTB2, act as IRES transacting factors of the HCV IRES.

The ASIP gene in the llama (Lama glama): Alternative transcripts, expression and relation with color phenotypes.

The Agouti gene (ASIP) is one of the most important genes for coat color determination in mammals. It has a complex structure with several promoters and alternative non-coding first exons that are transcribed into mRNAs with different 5'UTR. These mRNA isoforms regulate the temporal and spatial expression of the gene, producing diverse pigmentation patterns. Here, we studied ASIP transcriptional variants and their expression in the skin of llamas with different coat color phenotypes. We also described the ASIP locus, including promoter usage and the splicing events that originate each transcript variant. Using 5'RACE-PCR we isolated seven ASIP transcripts with alternative 5'UTR, where exons 1A, 1A', 1C, 1D, and a novel non-coding exon 1A" were identified. Additionally, new alternative spliced forms were found. The diversity of ASIP 5'UTRs is originated by a complex pattern of alternative promoter usage, multiple transcription start sites and splicing events that include exon skipping and alternative 3' splicing site selection. We found that ASIP was highly expressed in llamas with white and brown phenotypes while black animals presented very low expression. The main responsible for this difference was a fusion transcript between ASIP and NCOA6 genes, which was present in the skin of white and brown llamas but not in the black ones. The rest of ASIP transcripts presented very low expression in the skin, indicating that the main regulation point for ASIP gene expression is at the transcriptional level. Nevertheless, the characteristics of the 5'UTRs sequences suggest that alternative transcripts could be regulated differently at the protein synthesis level.

Insights on the SARS-CoV-2 genome variability: the lesson learned in Brazil and its impacts on the future of pandemics.

Since the beginning of the SARS-CoV-2 spread in Brazil, few studies have been published analysing the variability of viral genome. Herein, we described the dynamic of SARS-CoV-2 strains circulating in Brazil from May to September 2020, to better understand viral changes that may affect the ongoing pandemic. Our data demonstrate that some of the mutations identified are currently observed in variants of interest and variants of concern, and emphasize the importance of studying previous periods in order to comprehend the emergence of new variants. From 720 SARS-CoV-2 genome sequences, we found few sites under positive selection pressure, such as the D614G (98.5 %) in the spike, that has replaced the old variant; the V1167F in the spike (41 %), identified in the P.2 variant that emerged from Brazil during the period of analysis; and I292T (39 %) in the N protein. There were a few alterations in the UTRs, which was expected, however, our data suggest that the emergence of new variants was not influenced by mutations in UTR regions, since it maintained its conformational structure in most analysed sequences. In phylogenetic analysis, the spread of SARS-CoV-2 from the large urban centres to the countryside during these months could be explained by the flexibilization of social isolation measures and also could be associated with possible new waves of infection. These results allow a better understanding of SARS-CoV-2 strains that have circulated in Brazil, and thus, with relevant infomation, provide the potential viral changes that may have affected and/or contributed to the current and future scenario of the COVID-19 pandemic.

High-risk human papillomavirus-18 uses an mRNA sequence to synthesize oncoprotein E6 in tumors.

Cervical cancer is the fourth most common cause of cancer in women worldwide in terms of both incidence and mortality. Persistent infection with high-risk types of human papillomavirus (HPV), namely 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68, constitute a necessary cause for the development of cervical cancer. Viral oncoproteins E6 and E7 play central roles in the carcinogenic process by virtue of their interactions with cell master proteins such as p53, retinoblastoma (Rb), mammalian target of rapamycin (mTOR), and c-MYC. For the synthesis of E6 and E7, HPVs use a bicistronic messenger RNA (mRNA) that has been studied in cultured cells. Here, we report that in cervical tumors, HPV-18, -39, and -45 transcribe E6/E7 mRNAs with extremely short 5' untranslated regions (UTRs) or even lacking a 5' UTR (i.e., zero to three nucleotides long) to express E6. We show that the translation of HPV-18 E6 cistron is regulated by the motif ACCaugGCGCG(C/A)UUU surrounding the AUG start codon, which we term Translation Initiation of Leaderless mRNAs (TILM). This motif is conserved in all HPV types of the phylogenetically coherent group forming genus alpha, species 7, which infect mucosal epithelia. We further show that the translation of HPV-18 E6 largely relies on the cap structure and eIF4E and eIF4AI, two key translation initiation factors linking translation and cancer but does not involve scanning. Our results support the notion that E6 forms the center of the positive oncogenic feedback loop node involving eIF4E, the mTOR cascade, and p53.

Wnt5a promotes hippocampal postsynaptic development and GluN2B-induced expression via the eIF2α HRI kinase.

Wnt signaling plays a key role in neurodevelopment and neuronal maturation. Specifically, Wnt5a stimulates postsynaptic assemblies, increases glutamatergic neurotransmission and, through calcium signaling, generates nitric oxide (NO). Trying to unveil the molecular pathway triggering these postsynaptic effects, we found that Wnt5a treatment induces a time-dependent increases in the length of the postsynaptic density (PSD), elicits novel synaptic contacts and facilitates F-actin flow both in in vitro and ex vivo models. These effects were partially abolished by the inhibition of the Heme-regulated eukaryotic initiation factor 2α (HRI) kinase, a kinase which phosphorylates the initiation translational factor eIF2α. When phosphorylated, eIF2α normally avoids the translation of proteins not needed during stress conditions, in order to avoid unnecessary energetic expenses. However, phosphorylated eIF2α promotes the translation of some proteins with more than one open reading frame in its 5' untranslated region. One of these proteins targeted by Wnt-HRI-eIF2α mediated translation is the GluN2B subunit of the NMDA receptor. The identified increase in GluN2B expression correlated with increased NMDA receptor function. Considering that NMDA receptors are crucial for excitatory synaptic transmission, the molecular pathway described here contributes to the understanding of the fast and plastic translational mechanisms activated during learning and memory processes.

Characterization of the regulatory 5'-flanking region of bovine mucin 2 (MUC2) gene.

Throughout the intestinal epithelium surface there is an intricate polymer network composed by gel-forming mucins, which plays a protective role due to the formation of a physical, chemical and immunological barrier between the organism and the environment. Mucin 2 (MUC2) is the main mucin in the small and large intestine, and it is expressed specifically in the gastrointestinal tract (GIT), which makes its promoter region an important candidate for expression of heterologous genes of biotechnological interest in the GIT of bovine and other ruminants. In order to characterize the bovine MUC2 promoter we designed primers to amplify and isolate a candidate region for this promoter. The amplified sequence was confirmed by sequencing and cloned into a plasmid vector containing the luciferase (LUC) reporter gene. The regulatory sites of the MUC2 promoter already described in the literature were used to find the putative regulatory sites in the bovine MUC2 promoter region. With these data, some deletions were performed in order to find the promoter sequence with greatest expression capacity and specificity. The constructions were tested by transient transfection assays in LoVo cells (human colorectal adenocarcinoma) and bovine fibroblasts. The quantification of the relative expression of the promoter was measured using dual-luciferase assays. Real-time PCR was performed to analyze the expression of endogenous MUC2. The results presented herein prove that the isolated sequence corresponds to the promoter of bovine MUC2 gene, since it was able to induce expression of a reporter gene in an in vitro cell culture experimental platform.