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BBA - Gene Regulatory Mechanisms (v.1829, #5)

Editorial Board (pp. i).

Mouse Dazl and its novel splice variant functions in translational repression of target mRNAs in embryonic stem cells by Xingbo Xu; Xiaoying Tan; Qiong Lin; Bernhard Schmidt; Wolfgang Engel; D.V. Krishna Pantakani (pp. 425-435).

Dazl (deleted in azoospermia-like) is an RNA binding protein that is important for germ cell differentiation in vertebrates. In the present study, we report the identification of a novel Dazl isoform ( Dazl_Δ8) that results from alternative splicing of exon8 of mouse Dazl. We observed the expression of Dazl_Δ8 in various pluripotent cell types, but not in somatic cells. Furthermore, the Dazl_Δ8 splice variant was expressed along with the full-length isoform of Dazl ( Dazl_FL) throughout male germ-cell development and in the ovary. Sub-cellular localization studies of Dazl_Δ8 revealed a diffused cytoplasmic and large granular pattern, which is similar to the localization patterns of Dazl_FL protein. In contrast to the well documented translation stimulation function in germ cells, overexpression and downregulation studies of Dazl isoforms ( Dazl_FL and Dazl_Δ8) revealed a role for Dazl in the negative translational regulation of Mvh, a known target of Dazl, as well as Oct3/4 and Sox2 in embryonic stem cells (ESCs). In line with these observations, a luciferase reporter assay with the 3′UTRs of Oct3/4 and Mvh confirmed the translational repressive role of Dazl isoforms in ESCs but not in germ cells derived cell line GC-1. Further, we identified several putative target mRNAs of Dazl_FL and Dazl_Δ8 in ESCs through RNA-binding immunoprecipitation followed by whole genome transcriptome analysis. Collectively, our results show a translation repression function of Dazl in pluripotent stem cells.► We identified a novel splice variant of Dazl, i.e. Dazl_Δ8. ► Dazl_Δ8 showed spatiotemporal expression pattern similar to full length Dazl. ► Dazl functions in translational repression of target mRNAs in ES cells. ► We identified several putative target mRNAs for both Dazl isoforms.

Keywords: Abbreviations; maGSCs; multipotent adult germline stem cells; EGCs; embryonic germ cells; iPSCs; induced pluripotent stem cells; ECCs; embryonic carcinoma cells; GC-1; a spermatogonia cell line; RIP; RNA-binding protein immunoprecipitationESCs; Dazl; Splice variant; Translation repression; RNA binding protein

The role of microRNAs in hepatocyte nuclear factor-4alpha expression and transactivation by Zhongyan Wang; Peter A. Burke (pp. 436-442).

Hepatocyte nuclear factor (HNF)-4α is a key member of the transcription factor network regulating hepatocyte differentiation and function. Genetic and molecular evidence suggests that expression of HNF-4α is mainly regulated at the transcriptional level. Activation of HNF-4A gene involves the interaction of distinct sets of transcription factors and co-transcription factors within enhancer and promoter regions. Here we study the inhibitory effect of microRNAs (miRNAs) on the 3′-untranslated region (3′-UTR) of HNF-4A mRNA. The potential recognition elements of a set of miRNAs were identified utilizing bioinformatics analysis. The family members of miR-34 and miR-449, including miR-34a, miR-34c-5p and miR-449a, share the same target elements located at two distinct locations within the 3′-UTR of HNF-4A. The over-expression of miR-34a, miR-34c-5p or miR-449a in HepG2 cells led to a significant decrease in the activity of luciferase reporter carrying 3′-UTR of HNF-4A. The repressive effect on reporter activity was partially or fully eliminated when one or two of the binding site(s) for miR-34a/miR-34c-5p/miR-449a were deleted within the 3′-UTR. The protein level of HNF-4α was dramatically reduced by over-expression of miR-34a, miR-34c-5p and miR-449a, which correlates with a decrease in the binding activity of HNF-4α and transactivation of HNF-4α target genes. These results suggest that the recognition sites of miR-34a, miR-34c-5p and miR-449a within 3′-UTR of HNF-4A are functional. The mechanism of down-regulation of the binding activity and transactivation of HNF-4α by the miRNAs involves the decrease in HNF-4α protein level via miRNAs selectively targeting HNF-4A 3′-UTR, leading to the translational repression of HNF-4α expression.► HNF-4 plays a crucial role in the development and maintenance the liver phenotype. ► Specific miRNAs down-regulate HNF-4 expression by targeting 3′-UTR of HNF-4 mRNA. ► The miRNA dependent regulation affects the transactivation of HNF-4 target genes. ► The regulatory mechanism of miRNAs may have important impacts on liver phenotype.

Keywords: Abbreviations; HNF-4; hepatocyte nuclear factor (HNF)-4; miRNA; microRNA; 3′-UTR; 3′-untranslated region; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; EMSA; electrophoretic mobility shift assays; TTR; transthyretin; ApoB; apolipoprotein B; α1-AT; α1-antitrypsinHNF-4; MicroRNA; 3′-untranslated region; DNA binding activity; HepG2

Regulation of molecular chaperones through post-translational modifications: Decrypting the chaperone code by Philippe Cloutier; Benoit Coulombe (pp. 443-454).

Molecular chaperones and their associated cofactors form a group of highly specialized proteins that orchestrate the folding and unfolding of other proteins and the assembly and disassembly of protein complexes. Chaperones are found in all cell types and organisms, and their activity must be tightly regulated to maintain normal cell function. Indeed, deregulation of protein folding and protein complex assembly is the cause of various human diseases. Here, we present the results of an extensive review of the literature revealing that the post-translational modification (PTM) of chaperones has been selected during evolution as an efficient mean to regulate the activity and specificity of these key proteins. Because the addition and reciprocal removal of chemical groups can be triggered very rapidly, this mechanism provides an efficient switch to precisely regulate the activity of chaperones on specific substrates. The large number of PTMs detected in chaperones suggests that a combinatory code is at play to regulate function, activity, localization, and substrate specificity for this group of biologically important proteins. This review surveys the core information currently available as a starting point toward the more ambitious endeavor of deciphering the “chaperone code”.•Hsp70, Hsp90 and VCP are regulated by numerous post-translational modifications.•These PTMs form a code that orchestrates functional organization of the proteome.•The chaperone code may provide therapeutic strategies in chaperone diseases.

Keywords: Molecular chaperone; Post-translational modification; Hsp70; Hsp90; VCP

Transfection of siRNAs can alter miRNA levels and trigger non-specific protein degradation in mammalian cells by Xue-hai Liang; Christopher E. Hart; Stanley T. Crooke (pp. 455-468).

Sequence-non-specific effects of siRNAs that alter the expression of non-targeted genes have been reported, including competition of siRNAs with endogenous RISC components. However, the detailed mechanisms and subsequent effects of such competition are not well documented. Here we analyze the competition of miRNAs in mammalian cells with low concentrations of siRNAs, and found that: 1) transfection of different siRNAs in the low nanomolar range used to deplete target RNAs can reduce the levels of miRNAs in different cell types, 2) siRNA transfection results in rapid reduction of Ago2-associated miRNAs concurrent with accumulation of Ago2-bound siRNAs and a significant change in the expression levels of many miRNAs, 3) competition largely depends on Ago2 and not Dicer, 4) microarray analysis showed that the majority of highly expressed miRNAs are reduced, in a siRNA concentration dependent manner, and low abundant miRNAs may be unchanged or repressed and a few miRNAs appear to have increased levels, and 5) consistent with previous studies, the expression levels of mRNAs that are targeted by highly repressed miRNAs are preferentially increased. As a consequence of such competition, we observed that α-tubulin, a substrate of two up-regulated proteases, granzyme B and granzyme M, was rapidly degraded at the protein level upon siRNA transfection. Our results support a model in which transfection of siRNAs can change the levels of many miRNAs by competition for Ago2, leading to altered expression of many miRNA target genes, which can in turn affect downstream gene expression even at the protein level.► siRNAs can compete with miRNAs for Ago2 at concentrations typically used. ► Competition causes a significant change in the levels of many miRNAs. ► The effects of altered miRNA levels are unpredictable and can be profound. ► An example of such consequence is rapid degradation of α-tubulin at protein level.

Keywords: Abbreviations; RISC; RNA induced silencing complex; MEF; mouse embryonic fibroblast; ASO; antisense oligonucleotidessiRNA; RISC; Ago2; miRNA; Tubulin; Degradation

KSRP silencing favors neural differentiation of P19 teratocarcinoma cells by Matteo Giovarelli; Gabriele Bucci; Michela Pasero; Roberto Gherzi; Paola Briata (pp. 469-479).

Understanding the molecular mechanisms that control the balance between multipotency and differentiation is of great importance to elucidate the genesis of both developmental disorders and cell transformation events. To investigate the role of the RNA binding protein KSRP in controlling neural differentiation, we used the P19 embryonal carcinoma cell line that is able to differentiate into neuron-like cells under appropriate culture conditions. We have recently reported that KSRP controls the differentiative fate of multipotent mesenchymal cells owing to its ability to promote decay of unstable transcripts and to favor maturation of selected micro-RNAs (miRNAs) from precursors. Here we report that KSRP silencing in P19 cells favors neural differentiation increasing the expression of neuronal markers. Further, the expression of two master transcriptional regulators of neurogenesis, ASCL1 and JMJD3, was enhanced while the maturation of miR-200 family members from precursors was impaired in KSRP knockdown cells. These molecular changes can contribute to the reshaping of P19 cells transcriptome that follows KSRP silencing. Our data suggests that KSRP function is required to maintain P19 cells in a multipotent undifferentiated state and that its inactivation can orient cells towards neural differentiation.► KSRP function is required to keep teratocarcinoma P19 cells in an undifferentiated state. ► KSRP favors the expression of two master regulators of neural differentiation. ► KSRP promotes the maturation of miR-200 family members from precursors. ► Maturation of miR-200 family members is reduced during P19 cell neural differentiation. ► KSRP silencing in P19 cells promotes the reshaping of their transcriptome.

Keywords: mRNA degradation; microRNA maturation; RNA-binding protein; Neural differentiation

Retinoic acid impairs estrogen signaling in breast cancer cells by interfering with activation of LSD1 via PKA by Maria Neve Ombra; Annalisa Di Santi; Ciro Abbondanza; Antimo Migliaccio; Enrico Vittorio Avvedimento; Bruno Perillo (pp. 480-486).

More than 70% of breast cancers in women require estrogens for cell proliferation and survival. 17β-estradiol (E2) effect on mammary target cells is almost exclusively mediated by its binding to the estrogen receptor-α (ERα) that joins chromatin where it assembles active transcription complexes. The proliferative and pro-survival action of estrogens is antagonized in most cases by retinoic acid (RA), even though the cognate retinoic acid receptor-α (RARα) cooperates with ERα on promoters of estrogen-responsive genes.We have examined at the molecular level the crosstalk between these nuclear receptors from the point of view of their control of cell growth and show here that RA reverts estrogen-stimulated transcription of the pivotal anti-apoptotic bcl-2 gene by preventing demethylation of dimethyl lysine 9 in histone H3 (HeK9me2). As we previously reported, this is obtained by means of E2-triggered activation of the lysine-specific demethylase 1 (LSD1), an enzyme that manages chromatin plasticity in order to allow specific movements of chromosomal regions within the nucleus. We find that E2 fuels LSD1 by inducing migration of the catalytic subunit of protein kinase A (PKA) into the nucleus, where it targets estrogen-responsive loci. RA rescues LSD1-dependent disappearance of H3K9me2 at bcl-2 regulatory regions upon the prevention of PKA assembly to the same sites.•After E2 challenge, ERα and RARα join bcl-2 promoter with demethylation of H3K9me2.•E2-dependent demethylation of H3K9me2 is triggered by induction of LSD1 activity.•LSD1 activation by estrogens is mediated by the E2-induced increase of PKA signaling.•Concomitant addition of RA with E2 specifically prevents demethylation of H3K9me2.•RA blocks H3K9me2 demethylation by inhibiting PKA targeting to bcl-2 EREs.

Keywords: Nuclear receptor; Transcription regulation; Estrogen; Retinoic acid; Gene looping; Histone demethylation

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BBA - Gene Regulatory Mechanisms (v.1829, #5)