Untranslated regions of brain-derived neurotrophic factor mRNA control its translatability and subcellular localization.

Brain-derived neurotrophic factor (BDNF) promotes neuronal survival and growth during development. In the adult nervous system, BDNF is important for synaptic function in several biological processes such as memory formation and food intake. In addition, BDNF has been implicated in development and maintenance of the cardiovascular system. The Bdnf gene comprises several alternative untranslated 5' exons and two variants of 3' UTRs. The effects of these entire alternative UTRs on translatability have not been established. Using reporter and translating ribosome affinity purification analyses, we show that prevalent Bdnf 5' UTRs, but not 3' UTRs, exert a repressive effect on translation. However, contrary to previous reports, we do not detect a significant effect of neuronal activity on BDNF translation. In vivo analysis via knock-in conditional replacement of Bdnf 3' UTR by bovine growth hormone 3' UTR reveals that Bdnf 3' UTR is required for efficient Bdnf mRNA and BDNF protein production in the brain, but acts in an inhibitory manner in lung and heart. Finally, we show that Bdnf mRNA is enriched in rat brain synaptoneurosomes, with higher enrichment detected for exon I-containing transcripts. In conclusion, these results uncover two novel aspects in understanding the function of Bdnf UTRs. First, the long Bdnf 3' UTR does not repress BDNF expression in the brain. Second, exon I-derived 5' UTR has a distinct role in subcellular targeting of Bdnf mRNA.

Analysis of the Function of the Lymphocytic Choriomeningitis Virus S Segment Untranslated Region on Growth Capacity In Vitro and on Virulence In Vivo.

Lymphocytic choriomeningitis virus (LCMV) is a prototypic arenavirus. The function of untranslated regions (UTRs) of the LCMV genome has not been well studied except for the extreme 19 nucleotide residues of both the 5' and 3' termini. There are internal UTRs composed of 58 and 41 nucleotide residues in the 5' and 3' UTRs, respectively, in the LCMV S segment. Their functional roles have yet to be elucidated. In this study, reverse genetics and minigenome systems were established for LCMV strain WE and the function of these regions were analyzed. It was revealed that nucleotides 20-40 and 20-38 located downstream of the 19 nucleotides in the 5' and 3' termini, respectively, were involved in viral genome replication and transcription. Furthermore, it was revealed that the other internal UTRs (nucleotides 41-77 and 39-60 in the 5' and 3' termini, respectively) in the S segment were involved in virulence in vivo, even though these regions did not affect viral growth capacity in Vero cells. The introduction of LCMV with mutations in these regions attenuates the virus and may enable the production of LCMV vaccine candidates.

Cell Cycle Regulation by Alternative Polyadenylation of CCND1.

Global shortening of 3'UTRs by alternative polyadenylation (APA) has been observed in cancer cells. However, the role of APA in cancer remains unknown. CCND1 is a proto-oncogene that regulates progression through the G1-S phase of the cell cycle; moreover, it has been observed to be switching to proximal APA sites in cancer cells. To investigate the biological function of the APA of CCND1, we edited the weak poly(A) signal (PAS) of the proximal APA site to a canonical PAS using the CRISPR/Cas9 method, which can force the cells to use a proximal APA site. Cell cycle profiling and proliferation assays revealed that the proximal APA sites of CCND1 accelerated the cell cycle and promoted cell proliferation, but UTR-APA and CR-APA act via different molecular mechanisms. These results indicate that PAS editing with CRISPR/Cas9 provides a good method by which to study the biological function of APA.

Dual function of a cis-acting RNA element that acts as a replication enhancer and a translation repressor in a plant positive-stranded RNA virus.

The genome of red clover necrotic mosaic virus is divided into two positive-stranded RNA molecules of RNA1 and RNA2, which have no 5' cap structure and no 3' poly(A) tail. Previously, we showed that any mutations in the cis-acting RNA replication elements of RNA2 abolished its cap-independent translational activity, suggesting a strong link between RNA replication and translation. Here, we investigated the functions of the 5' untranslated region (UTR) of RNA2 and revealed that the basal stem-structure (5'BS) predicted in the 5' UTR is essential for robust RNA replication. Interestingly, RNA2 mutants with substitution or deletion in the right side of the 5'BS showed strong translational activity, despite their impaired replication competency. Furthermore, nucleotide sequences other than the 5'BS of the 5' UTR were essential to facilitate the replication-associated translation. Overall, these cis-acting RNA elements seem to coordinately regulate the balance between RNA replication and replication-associated translation.

Overexpression of miR-484 and miR-744 in Vero cells alters Dengue virus replication

BACKGROUND Dengue is considered one of the world’s most important mosquito-borne diseases. MicroRNAs (miRNAs) are small non-coding single-stranded RNAs that play an important role in the regulation of gene expression in eukaryotes. Although miRNAs possess antiviral activity against many mammalian-infecting viruses, their involvement in Dengue virus (DENV) replication remains poorly understood. OBJECTIVE To determine the role of miR-484 and miR-744 in DENV infection and to examine whether DENV infection alters the expression of both miRNAs. METHODS We used bioinformatics tools to explore the relationship between DENV and cellular miRNAs. We then overexpressed miR-484 or miR-744 in Vero cells to examine their role in DENV replication using flow cytometry, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), and western blotting. FINDINGS We found several cellular miRNAs that target a conserved region within the 3′ untranslated region (3′ UTR) of the genome of the four DENV serotypes and found that overexpression of miR-484 or miR-744 inhibits infection by DENV-1 to DENV-4. Furthermore, we observed that DENV RNA might be involved in the downregulation of endogenous miR-484 and miR-744. CONCLUSION Our study identifies miR-484 and miR-744 as two possible restriction host factors against DENV infection. However, further studies are needed to directly verify whether miR-484 and miR-744 both have an anti-DENV effect in vivo.

Implication of CA repeated tracts on post-transcriptional regulation in Trypanosoma cruzi.

In Trypanosoma cruzi gene expression regulation mainly relays on post-transcriptional events. Nevertheless, little is known about the signals which control mRNA abundance and functionality. We have previously found that CA repeated tracts (polyCA) are abundant in the vicinity of open reading frames and constitute specific targets for single stranded binding proteins from T. cruzi epimastigote. Given the reported examples of the involvement of polyCA motifs in gene expression regulation, we decided to further study their role in T. cruzi. Using an in silico genome-wide analysis, we identify the genes that contain polyCA within their predicted UTRs. We found that about 10% of T. cruzi genes carry polyCA therein. Strikingly, they are frequently concurrent with GT repeated tracts (polyGT), favoring the formation of a secondary structure exhibiting the complementary polydinucleotides in a double stranded helix. This feature is found in the species-specific family of genes coding for mucine associated proteins (MASPs) and other genes. For those polyCA-containing UTRs that lack polyGT, the polyCA is mainly predicted to adopt a single stranded structure. We further analyzed the functional role of such element using a reporter approach in T. cruzi. We found out that the insertion of polyCA at the 3' UTR of a reporter gene in the pTEX vector modulates its expression along the parasite's life cycle. While no significant change of the mRNA steady state of the reporter gene could be detected at the trypomastigote stage, significant increase in the epimastigote and reduction in the amastigote stage were observed. Altogether, these results suggest the involvement of polyCA as a signal in gene expression regulation in T. cruzi.

Genetic complexity of the human surfactant-associated proteins SP-A1 and SP-A2.

Pulmonary surfactant protein A (SP-A) plays a key role in innate lung host defense, in surfactant-related functions, and in parturition. In the course of evolution, the genetic complexity of SP-A has increased, particularly in the regulatory regions (i.e. promoter, untranslated regions). Although most species have a single SP-A gene, two genes encode SP-A in humans and primates (SFTPA1 and SFTPA2). This may account for the multiple functions attributed to human SP-A, as well as the regulatory complexity of its expression by a relatively diverse set of protein and non-protein cellular factors. The interplay between enhancer cis-acting DNA sequences and trans-acting proteins that recognize these DNA elements is essential for gene regulation, primarily at the transcription initiation level. Furthermore, regulation at the mRNA level is essential to ensure proper physiological levels of SP-A under different conditions. To date, numerous studies have shown significant complexity of the regulation of SP-A expression at different levels, including transcription, splicing, mRNA decay, and translation. A number of trans-acting factors have also been described to play a role in the control of SP-A expression. The aim of this report is to describe the genetic complexity of the SFTPA1 and SFTPA2 genes, as well as to review regulatory mechanisms that control SP-A expression in humans and other animal species.

Comparative transcriptomics of Eastern African cichlid fishes shows signs of positive selection and a large contribution of untranslated regions to genetic diversity.

The hundreds of endemic species of cichlid fishes in the East African Great Lakes Tanganyika, Malawi, and Victoria are a prime model system in evolutionary biology. With five genomes currently being sequenced, eastern African cichlids also represent a forthcoming genomic model for evolutionary studies of genotype-to-phenotype processes in adaptive radiations. Here we report the functional annotation and comparative analyses of transcriptome data sets for two eastern African cichlid species, Astatotilapia burtoni and Ophthalmotilapia ventralis, representatives of the modern haplochromines and ectodines, respectively. Nearly 647,000 expressed sequence tags were assembled in more than 46,000 contigs for each species using the 454 sequencing technology, largely expanding the current sequence data set publicly available for these cichlids. Total predicted coverage of their proteome diversity is approximately 50% for both species. Comparative qualitative and quantitative analyses show very similar transcriptome data for the two species in terms of both functional annotation and relative abundance of gene ontology terms expressed. Average genetic distance between species is 1.75% when all transcript types are considered including nonannotated sequences, 1.33% for annotated sequences only including untranslated regions, and decreases to nearly half, 0.95%, for coding sequences only, suggesting a large contribution of noncoding regions to their genetic diversity. Comparative analyses across the two species, tilapia and the outgroup medaka based on an overlapping data set of 1,216 genes (∼526 kb) demonstrate cichlid-specific signature of disruptive selection and provide a set of candidate genes that are putatively under positive selection. Overall, these data sets offer the genetic platform for future comparative analyses in light of the upcoming genomes for this taxonomic group.

Analysis of the GAD1 promoter: trans-acting factors and DNA methylation converge on the 5' untranslated region.

GAD67 corresponds to one of two enzymes that decarboxylates glutamate to produce γ-aminobutyric acid, the main inhibitory neurotransmitter in the mammalian central nervous system, hence defining the cellular phenotype of a diverse set of inhibitory interneurons of the brain. Reduced cortical GAD67 mRNA levels have consistently been reported in schizophrenia and bipolar disorder with psychosis. The human gene encoding GAD67, GAD1, is located on chromosome 2q31.1 and the transcriptional start site resides within a large CpG island that spans a region extending from upstream through the first exon. We have analyzed the GAD1 promoter using transient transfection analysis of upstream and downstream sequences in NT2 cells, a human neuroprogenitor cell line. Interestingly, results from these studies show that cis-acting regulatory elements are located downstream of the RNA start site and are in the region corresponding to the first exon. Trans-acting factors such as Pitx2 and the Dlx family of transcription factors are active in promoting downstream reporter expression even when all of the 5' flanking sequences are removed. However, those constructs that contain an internal deletion from +66 to +173 bp fail to support expression even when these factors are provided in trans. We have previously shown that the Class I histone deacetylase inhibitor MS-275 potently activates GAD1 mRNA expression in NT2 cells suggesting the possibility that the promoter is sensitive to drugs that induce chromatin remodeling. Using methyl DNA immuneprecipitation of MS-275-treated NT2 cells, we provide data showing that Class I HDAC inhibition mediated an increase in GAD1 expression and that this was accompanied by decreased GAD1 promoter methylation. Moreover, the reduced levels of GAD1 DNA methylation are highest in those regions proximal to the location of the in vitro defined cis-acting regulatory elements. Our data suggest that changes in promoter methylation associated with gene regulation are not random but overlap the locations of proximal cis-acting elements. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

MicroRNAs: control and loss of control in human physiology and disease.

Analysis of the human genome indicates that a large fraction of the genome sequences are RNAs that do not encode any proteins, also known as non-coding RNAs. MicroRNAs (miRNAs) are a group of small non-coding RNA molecules 20-22 nucleotides (nt) in length that are predicted to control the activity of approximately 30% of all protein-coding genes in mammals. miRNAs play important roles in many diseases, including cancer, cardiovascular disease, and immune disorders. The expression of miRNAs can be regulated by epigenetic modification, DNA copy number change, and genetic mutations. miRNAs can serve as a valuable therapeutic target for a large number of diseases. For miRNAs with oncogenic capabilities, potential therapies include miRNA silencing, antisense blocking, and miRNA modifications. For miRNAs with tumor suppression functions, overexpression of those miRNAs might be a useful strategy to inhibit tumor growth. In this review, we discuss the current progress of miRNA research, regulation of miRNA expression, prediction of miRNA targets, and regulatory role of miRNAs in human physiology and diseases, with a specific focus on miRNAs in pancreatic cancer, liver cancer, colorectal cancer, cardiovascular disease, the immune system, and infectious disease. This review provides valuable information for clinicians and researchers who want to recognize the newest advances in this new field and identify possible lines of investigation in miRNAs as important mediators in human physiology and diseases.

Engineering eukaryotic protein factories.

The biopharmaceuticals market is currently outperforming the pharmaceuticals market and is now valued at US$ 48 billion with an average annual growth of 19%. Behind this success is a 100-fold increase in productivities of eukaryotic expression systems. However, the productivity per cell has remained unchanged for more than 10 years. The engineering of the ER-resident protein folding machinery is discussed together with an overview of signal transduction pathways activated by heterologous protein overexpression to increase cell specific productivities.

[Reverse genetics system for measles virus: establishment and applications for analysis of virus replication and pathogenesis].

In 1990 Kobune et al. succeeded in isolating pathogenic wild-type strains of measles virus (MV) using a marmoset B-lymphoblastoid cell line B95a. Their data indicated that MV strains that have been used in laboratories are attenuated strains through the adaptation to grow in a variety of cultured cells. We have established a very efficient reverse genetics system that allows us to engineer the genome of a wild-type MV strain at will by site-directed mutagenesis or recombination. Using the system it was shown that (1) the H protein determines tropism of MV, (2) the M protein regulates mode of MV spread, (3) the C protein inhibits host innate immune responses, and (4) the long untranslated regions in the M and F genes function to moderate cytopathogenicity by MV. These data advanced our understanding of molecular bases for MV pathogenicity and mechanisms of MV adaptation to grow in cultured cells.

Analyses of murine postsynaptic density-95 identify novel isoforms and potential translational control elements.

Postsynaptic density-95 (PSD-95) is an evolutionarily conserved synaptic adaptor protein that is known to bind many proteins including the NMDA receptor. This observation has implicated it in many NMDA receptor-dependent processes including spatial learning and synaptic plasticity. We have cloned and characterised the murine PSD-95 gene. In addition, we have identified two previously uncharacterised splice variants of the major murine PSD-95 transcript (PSD-95alpha): PSD-95alpha-2b results from an extension of exon 2 and PSD-95alpha-Delta18 from the temporal exclusion of exon 18. The presence of PSD-95alpha-2b sequences in other PSD-95 family members implicates this peptide stretch as functionally significant. Another potential transcript (PSD-95gamma) was also identified based on examination of EST databases. Immunoprecipitation assays demonstrate that proteins corresponding in size to PSD-95alpha-Delta18 and PSD-95gamma interact with the NMDA receptor, suggesting an important biological role for these isoforms. Finally, we have performed bioinformatics analyses of the PSD-95 mRNA untranslated regions, identifying multiple translational control elements that suggest protein production could be regulated post-transcriptionally. The variety of mRNA isoforms and regulatory elements identified provides for a high degree of diversity in the structure and function of PSD-95 proteins.

Evolution and functional classification of vertebrate gene deserts.

Large tracts of the human genome, known as gene deserts, are devoid of protein-coding genes. Dichotomy in their level of conservation with chicken separates these regions into two distinct categories, stable and variable. The separation is not caused by differences in rates of neutral evolution but instead appears to be related to different biological functions of stable and variable gene deserts in the human genome. Gene Ontology categories of the adjacent genes are strongly biased toward transcriptional regulation and development for the stable gene deserts, and toward distinctively different functions for the variable gene deserts. Stable gene deserts resist chromosomal rearrangements and appear to harbor multiple distant regulatory elements physically linked to their neighboring genes, with the linearity of conservation invariant throughout vertebrate evolution.

Untranslated regions from C4 amaranth AhRbcS1 mRNAs confer translational enhancement and preferential bundle sheath cell expression in transgenic C4 Flaveria bidentis.

Many aspects of photosynthetic gene expression are posttranscriptionally regulated in C4 plants. To determine if RbcS mRNA untranslated regions (UTRs) in themselves could confer any characteristic C4 expression patterns, 5'- and 3'-UTRs of AhRbcS1 mRNA from the C4 dicot amaranth were linked to a gusA reporter gene. These were constitutively transcribed from a cauliflower mosaic virus promoter and assayed for posttranscriptional expression patterns in transgenic lines of the C4 dicot Flaveria bidentis. Three characteristic C4 expression patterns were conferred by heterologous AhRbcS1 UTRs in transgenic F. bidentis. First, the AhRbcS1 UTRs conferred strong translational enhancement of gusA expression, relative to control constructs lacking these UTRs. Second, while the UTRs did not appear to confer tissue-specific expression when analyzed by beta-glucuronidase activity assays, differences in gusA mRNA accumulation were observed in leaves, stems, and roots. Third, the AhRbcS1 UTRs conferred preferential gusA expression (enzyme activity and gusA mRNA accumulation) in leaf bundle sheath cells. AhRbcS1 UTR-mediated translational enhancement was also observed in transgenic C3 plants (tobacco [Nicotiana tabacum]) and in in vitro translation extracts. These mRNAs appear to be translated with different efficiencies in C4 versus C3 plants, indicating that processes determining overall translational efficiency may vary between these two categories of higher plants. Our findings suggest that the AhRbcS1 5'-UTR functions as a strong translational enhancer in leaves and other tissues, and may work synergistically with the 3'-UTR to modulate overall levels of Rubisco gene expression in different tissues and cell types of C4 plants.

The short 5' untranslated region of the betaA3/A1-crystallin mRNA is responsible for leaky ribosomal scanning.

Leaky ribosomal scanning allows the expression of multiple proteins from a single mRNA by occasionally skipping the first start codon, and initiating translation at a subsequent one. BetaA3- and betaA1-crystallin, two members of the beta-crystallin family of vertebrate eye lens proteins, are produced via this mechanism, of which, until now, only very few examples have been found in eukaryotic genes. Since the two start codons on the betaA3/A1 messenger lie in the same reading frame, the two translated proteins are identical, except for the 17 residues shorter N-terminal extension of betaA1-crystallin. It has been suggested that the very short leader (5-7 nucleotides) of the betaA3/A1 messenger might cause slippage at the first start codon, although the unfavorable context of this start codon might also be responsible. Using transient transfections, we now demonstrate that increasing the length of the leader sequence to 67 nucleotides indeed completely abolishes translation initiation at the second start codon, and thus expression of the betaA1-crystallin protein. Messengers having a leader of 5, 7 or 14 nucleotides all express both betaA3- and betaA1-crystallin at very similar relative levels.

SP-A 3'-UTR is involved in the glucocorticoid inhibition of human SP-A gene expression.

The synthetic glucocorticoid dexamethasone has a major inhibitory effect on human surfactant protein A1 (SP-A1) and SP-A2 gene expression that occurs at both the transcriptional and posttranscriptional levels. Toward the identification of cis-acting elements that may be involved in the dexamethasone regulation of SP-A mRNA stability, chimeric chloramphenicol acetyltransferase (CAT) constructs that contained various portions of SP-A1 or SP-A2 cDNA in place of the native CAT 3'-untranslated region (UTR) were transiently transfected into the lung adenocarcinoma cell line NCI-H441. CAT activity was reduced in NCI-H441 cells by exposure to 100 nM dexamethasone only for the chimeric CAT constructs that contained the SP-A 3'-UTR. Moreover, the inhibitory response seen with dexamethasone was greater for the 3'-UTR derived from the SP-A1 allele 6A3 than with the 3'-UTR derived from either the SP-A1 allele 6A2 or SP-A2 allele 1A0, indicating differential regulation between SP-A genes and/or alleles.