Alternative pre-mRNA splicing of the mu opioid receptor gene, OPRM1: Insight into complex mu opioid actions

Most opioid analgesics used clinically, including morphine and fentanyl, as well as the recreational drug heroin, act primarily through the mu opioid receptor, a class A Rhodopsin-like G protein-coupled receptor (GPCR). The single-copy mu opioid receptor gene

Therapeutic Hemoglobin Levels after Gene Transfer in β-Thalassemia Mice and in Hematopoietic Cells of β-Thalassemia and Sickle Cells Disease Patients

Preclinical and clinical studies demonstrate the feasibility of treating β-thalassemia and Sickle Cell Disease (SCD) by lentiviral-mediated transfer of the human β-globin gene. However, previous studies have not addressed whether the ability of lentiviral vectors to increase hemoglobin synthesis might vary in different patients

Functional polymorphisms of the brain serotonin synthesizing enzyme tryptophan hydroxylase-2

Many neuropsychiatric disorders are considered to be related to the dysregulation of brain serotonergic neurotransmission. Tryptophan hydroxylase-2 (TPH2) is the neuronal-specific enzyme that controls brain serotonin synthesis. There is growing genetic evidence for the possible involvement of TPH2 in serotonin-related neuropsychiatric disorders; however, the degree of genetic variation in TPH2 and, in particular, its possible functional consequences remain unknown. In this short review, we will summarize some recent findings with respect to the functional analysis of TPH2

U1 snRNP-Dependent Suppression of Polyadenylation: Physiological Role and Therapeutic Opportunities in Cancer

Pre-mRNA splicing and polyadenylation are critical steps in the maturation of eukaryotic mRNA. U1 snRNP is an essential component of the splicing machinery and participates in splice-site selection and spliceosome assembly by base-pairing to the 5′ splice site. U1 snRNP also plays an additional, nonsplicing global function in 3′ end mRNA processing; it actively suppresses the polyadenylation machinery from using early, mostly intronic polyadenylation signals which would lead to aberrant, truncated mRNAs. Thus, U1 snRNP safeguards pre-mRNA transcripts against premature polyadenylation and contributes to the regulation of alternative polyadenylation. Here, we review the role of U1 snRNP in 3′ end mRNA processing, outline the evidence that led to the recognition of its physiological, general role in inhibiting polyadenylation, and finally highlight the possibility of manipulating this U1 snRNP function for therapeutic purposes in cancer

Posttranscriptional Regulation and RNA Binding Proteins in Cancer Biology

EditorialFollowing the completion of the human genome sequence and the concomitant technological innovations required for whole genome analyses, the last decade has witnessed an explosion of data and information concerning the posttranscriptional regulation of gene expression, in both pathological and nonpathological contexts. Among the most notable posttranscriptional events studied are the widespread usage of alternative splicing, the pleiotropic regulatory roles of miRNAs, and breakthroughs in the understanding of the control of gene expression by noncoding RNA transcripts. In this special issue of this journal, the spotlight is centered on the role that various mechanisms of posttranscriptional regulation—and the RNA binding proteins (RBPs) that control them—play in cancer biology

5′ UTR Control of Native ERG and of Tmprss2:ERG Variants Activity in Prostate Cancer

<div><p>ERG, a member of the ETS transcription factor family, is frequently overexpressed in prostate cancer as a result of its fusion to the androgen-responsive Tmprss2 gene. Different genomic rearrangements and alternative splicing events around the junction region lead to multiple combination of Tmprss2:ERG fusion transcripts that correlate with different tumor aggressiveness, but their specific functions and biological activities are still unclear. The complexity of ERG expression pattern is compounded by the use of alternative promoters, splice sites, polyadenylation sites and translation initiation sites in both the native and fusion contexts. Our systematic characterization of native ERG and Tmprss2:ERG variants reveals that their different oncogenic potential is impacted by the status of the Ets domain and the configuration of the 5′ UTR region. In particular, expression and activity of functional ERG and Tmprss2:ERG variants are influenced both by translation initiation signals within the different isoforms and by inhibitory upstream Open Reading Frames (uORF) in their 5′ UTRs. Stable expression of ERG and Tmprss2:ERG variants promoted cell migration/invasion, induced a block of proliferation and induced a senescence-like state, suggesting a role for these variants in the prostate tumorigenesis process. In addition to Tmprss2:ERG fusion products, a group of related native ERG isoforms is also highly over-expressed in fusion-carrying prostate cancers, and share the same translation initiation site (in ERG exon 4) with the commonly observed Tmprss2 exon1 joined to ERG exon 4 (T1:E4) fusion-derived variant. Usage of this ATG can be preferentially down-regulated by directed antisense-based compounds, possibly representing the basis of a targeted approach that distinguishes between tumor–associated and normal ERG.</p> </div

Role of the 5′ UTR region in ERG and Tmprss2:ERG variants expression.

<p>(<b>A</b>) The native 5′ UTR of ERG-1b, ERG-1c and T1:E4 were replaced with a common one from the expression vector (in orange), and an optimized Kozak sequence. The replaced ATGs are in exon 3 (M3), 1c (M1c) and 4 (M4a) (<b>B</b>) Context of multiple in-frame ATG used as start codons in various ERG and Tmprss2:ERG variants. The sequence around the ATG is aligned to a consensus Kozak sequence, with the important conserved positions at -3 (R) and +4 (G) highlighted in red (counting the A as +1). Context is evaluated as ‘strong’ (S) if both positions are conserved, and ‘weak’ (W) if they are not. An expanded analysis of ATGs in the 5′UTR region of ERG and Tmprss2:ERG is reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049721#pone.0049721.s004" target="_blank">Table S1</a>. (<b>C</b>) WB analysis of the variants and mutants represented in (A). Improving the M3 context favors its use at the expenses of the M4a ATG. (<b>D</b>) uORFs that might influence translation efficiency of ERG variants in the 5′ UTRs of ERG-1b and several common Tmprss2:ERG variants. Red ticks indicate ATGs, boxes below indicate the putative uORF generated and their approximate length (drawing not to scale). Red boxes indicate strong ATG contexts and green weak ones (as defined in (B)). More details about the uORFs are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049721#pone.0049721.s004" target="_blank">Table S1</a>. (<b>E</b>) Effect of independently mutating uORFs’ ATG in ERG-1b: mutation of the first ATG in exon 2 releases suppression of translation and increases levels of ERG from the ATG in exon 4. (<b>F</b>) Expression of ERG and Tmprss2:ERG variants<b>.</b> Full-length cDNAs for the variants, including their entire 5′ UTRs, were expressed and lysates from transiently transfected HeLa cells were analyzed by western blot.</p

Mapping of translation initiation of ERG isoforms.

<p>(<b>A</b>) N-terminal heterogeneity in human native ERG variants. The 5′ regions for the indicated variants are reproduced. Boxes represent exons with the ORF in blue. Red ticks below the exons indicate in-frame ATGs in exons 1c and exons 3 to 5. The out-of-frame products caused by exon skipping, from the otherwise in frame ATGs are indicated in red. (<b>B</b>) Western Blot (WB) of transient expression of the variants in HeLa cells. Antibody C20 recognizes exogenous protein and an endogenous band at ∼44 KDa corresponding in size to Δ4 variants. Antibody C17 preferentially recognizes exogenous ERG bands. (<b>C</b>) Mutations that independently eliminate the 3 in-frame ATGs in exon 4 were introduced into the cDNA of T1:E4 and analyzed as above. (<b>D</b>), Similarly, mutations were introduced to eliminate the ATGs in exon 3 (ERG-1b) or in exon 1c (ERG-1c) by themselves or in combination with mutations in the first in-frame ATG (M4a) in exon 4 (ERG-1b and ERG-1c).</p

Human ERG gene structure and main isoforms.

<p>(<b>A</b>) Top: The ∼300 Kb human ERG locus, drawn roughly to scale. Approximate intron sizes are indicated, along with exons position (bars). Red indicates first exons, blue common alternative ones and gray uncommon ones. Middle: Exon structure, with exon sizes at the bottom. Blue boxes indicate the main predicted ORFs, white boxes the untranslated regions and gray the uncommon exons. Red circles indicate polyA sites. Bottom: alignment of the exons forming the main ORF (ERG-1b) with the protein’s domains. Numbers indicate size in amino acids. PNT = pointed domain, AD = alternative domain, Ets = Ets domain, TAD = transactivation domain. Asterisk and circle indicate position of the first and second ATG. (<b>B</b>) Human ERG main variants. Alignment of exons forming the 30 main RNA variants of human ERG. Blue indicates the ORF, light blue the additional region from the ATG in exon 3. For each variant, the proposed name is indicated next to previous nomenclature (if available). The proposed protein name is reported along the predicted size in aa and KDa. Variants derived from the alternative usage of promoter 1a and 1b are paired as they lead to related mRNAs and identical proteins.</p

Differential ERG variant expression in normal tissues and PCa cells.

<p>Quantification by qPCR of alternative ERG variants in normal tissue (<b>A, D</b>), primary PCa samples (<b>B, E</b>) and PCa cell lines. (<b>C, F</b>). (<b>A–C</b>) Quantification of promoter usage. Primer sets specific for the 3 different ERG promoters and for the Tmprss2:ERG fusion where used, along with a primer set spanning exons 5–7 to quantify total ERG. Normal tissues do not express the fusion product. Expression of variants 1a and 1c is virtually undetectable in the PCa cell lines analyzed. For panel C, open symbols indicate LnCap, DU145 and C4-2 (not expressing Tmprss2:ERG fusion), full symbols indicate VCap and NCI-H660 (expressing Tmprss2:ERG fusion). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049721#pone.0049721.s001" target="_blank">Fig. S1</a> A–C. (<b>D–F</b>) Quantification by qPCR of alternative polyadenylation usage. Primer sets specific for the 3 different polyA sites where used, along with a primer set to quantify total ERG and one set to quantify total exon 11 levels in order to infer 11SpA usage. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049721#pone.0049721.s001" target="_blank">Fig. S1</a> D–F. In all cases, each indicated value represents averages of ≥3 independent experiments and is presented as ΔC(t) normalized to the housekeeping gene GAPDH, therefore a “high” ΔC(t) value means low levels of expression and a “low” value means high level of expression. Horizontal bars indicate the mean. Asterisks indicate that the product was not detected in the sample and would therefore be equivalent to an experimental point at the top of the table, but it is not reflected by the mean.</p