Post-transcriptional Regulatory Machinery Research Articles

3097 – RNA EDITOR-EXONUCLEASE AXIS SCULPTS THE TRANSCRIPTOME DURING TERMINAL ERYTHROID DIFFERENTIATION

Erythropoiesis is a committed process by which erythroid progenitors become mature red blood cells (RBCs). Reticulocytes are terminal-staged, immature RBCs which contain selected residual RNA after enucleation. Normally, reticulocytes are efficiently processed and typically represent a small percentage of cells in human peripheral blood. In contrast, when differentiated from iPS or CD34+ hematopoietic stem/progenitor cells in vitro, erythroid cells tend to arrest at the reticulocyte stage. This is the major bottleneck for the complete recapitulation of erythropoiesis in vitro, and a hindrance to modeling hemoglobinopathies in cell cultures. Recent studies have highlighted that uridylation by Terminal Uridylyl Transferases (TUTases 7/4, official names are ZCCHC6/11) occurs on a broad spectrum of RNA classes in mammalian cells. Oligo-uridylated RNA is then recognized by exoribonucleases and targeted for decay. We posited that this is the machinery behind RNA degradation that accompanies terminal erythropoiesis. Utilizing both constitutional and erythroid-specific conditional murine knockout models, we found that blood from TUTase Zcchc6 RNA editor knockout embryos indeed exhibit reticulocytosis with a terminal maturation defect, as documented by FACS, histology, and hematological profiling. Murine strains deficient in the downstream exonuclease, Dis3l2, phenocopied the RNA decay defect of the Zcchc6 KO. Furthermore, knockout models did not show signs of hemolysis suggesting a cell intrinsic and niche-independent role for the TUTase7-Dis3l2 axis in promoting red blood cell maturation. Modulating the expression of this axis holds promise for enhancing in vitro erythropoiesis and modeling of hemoglobinopathies. Future mechanism studies will also reveal a post-transcriptional regulatory machinery governing maturation from reticulocytes to erythrocytes.

Developing a Versatile Shotgun Cloning Strategy for Single-Vector-Based Multiplex Expression of Short Interfering RNAs (siRNAs) in Mammalian Cells.

As an important post-transcriptional regulatory machinery mediated by ∼21nt short-interfering double-stranded RNA (siRNA), RNA interference (RNAi) is a powerful tool to delineate gene functions and develop therapeutics. However, effective RNAi-mediated silencing requires multiple siRNAs for given genes, a time-consuming process to accomplish. Here, we developed a user-friendly system for single-vector-based multiplex siRNA expression by exploiting the unique feature of restriction endonuclease BstXI. Specifically, we engineered a BstXI-based shotgun cloning (BSG) system, which consists of three entry vectors with siRNA expression units (SiEUs) flanked with distinct BstXI sites, and a retroviral destination vector for shotgun SiEU assembly. For proof-of-principle studies, we constructed multiplex siRNA vectors silencing β-catenin and/or Smad4 and assessed their functionalities in mesenchymal stem cells (MSCs). Pooled siRNA cassettes were effectively inserted into respective entry vectors in one-step, and shotgun seamless assembly of pooled BstXI-digested SiEU fragments into a retroviral destination vector followed. We found these multiplex siRNAs effectively silenced β-catenin and/or Smad4, and inhibited Wnt3A- or BMP9-specific reporters and downstream target expression in MSCs. Furthermore, multiplex silencing of β-catenin and/or Smad4 diminished Wnt3A and/or BMP9-induced osteogenic differentiation. Collectively, the BSG system is a user-friendly technology for single-vector-based multiplex siRNA expression to study gene functions and develop experimental therapeutics.

RNA 'Information Warfare' in Pathogenic and Mutualistic Interactions.

Regulatory non-coding RNAs are emerging as key players in host-pathogen interactions. Small RNAs such as microRNAs are implicated in regulating plant transcripts involved in immunity and defence. Surprisingly, RNAs with silencing properties can be translocated from plant hosts to various invading pathogens and pests. Small RNAs are now confirmed virulence factors, with the first report of fungal RNAs that travel to host cells and hijack post-transcriptional regulatory machinery to suppress host defence. Here, we argue that trans-organism movement of RNAs represents a common mechanism of control in diverse interactions between plants and other eukaryotes. We suggest that extracellular vesicles are the key to such RNA movement events. Plant pathosystems serve as excellent experimental models to dissect RNA 'information warfare' and other RNA-mediated interactions.

Natural killer cells expanded on membrane-bound IL15 or IL21 activate different miRNA transcriptional profiles with functional consequences

MicroRNA (miRNA) are non-coding regulatory RNA that are key posttranscriptional regulatory machinery of cells. miRNA play important role in several physiological functions during embryonic development, differentiation, cancer progression as well as hematopoiesis, and regulation of immune responses. miRNA regulate development of various immune cells including T and Natural killer (NK) cells. Owing to significant interest in adoptive immunotherapy, numerous methods for expanding NK cells have been developed in recent years. We have previously developed a robust platform for ex-vivo expansion of human NK cells using K562 expressing membrane-bound IL21 (K562.mbIL21). The study of the role of miRNA in NK cells biology is an emerging area of research in development, homeostasis and immune responses. However, the role and effect of expansion of NK cells on miRNA and consequent impact on post-transcriptional regulation of protein expression in expanded NK cells remain to be studied. In this study we investigated the role of distinctive miRNA expression profiles that could influence post-transcriptional regulation of gene expression, and consequently differential protein expression in NK cells expanded on K562.mbIL21 platform compared to primary unexpanded NK cells. We had previously shown that K562.mbIL21 platform enables significantly higher expansion of NK cells compared to K562 expressing membrane bound IL15 (K562 mbIL15), therefore in this study we also compared miRNA expression profiles of NK cells expanded on K562.mbIL15 and K562.mbIL21. We used primary protein and miRNA obtained from, human NK cells derived from 4 healthy donors, Pre and post-expansion on K562.mbIL15 and K562.mbIL21 platforms. miRNA expression profiles were analyzed using Nanostring's nCounter. Initial screening of our dataset revealed 13 miRNAs that are differentially expressed by at least 16 folds between fresh and expanded NK cells or mbIL15 and mbIL21. Comparison between the mbIL15 and mbIL21 expanded cells revealed differential expression of miRNAs critical for regulation of cell cycle proteins as well as key cytokines (TNF-a and interferon-g) such as miR-124-3p and miR 146a-5p (76x and 32x lower in mbIL21 expanded cells respectively). Also, analysis of protein expression by RPPA revealed upregulation of cell cycle proteins cyclin D1 and AMPK, and downregulation of pro-apoptotic protein Bim in mbIL21 expanded NK cells compared with mbIL15 and/or primary unexpanded cells. We are currently correlating miRNA expression and proteomic profiles of expanded and unexpanded cells to identify possible targets of miRNA regulation and subsequently study specific roles of these miRNA in this expansion system by overexpression o/blockade of target miRNA.

Microarray to deep sequencing: transcriptome and miRNA profiling to elucidate molecular pathways in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical manifestations and autoantibody repertoires. The etiology of SLE is multifactorial involving genetic, epigenetic and environmental factors. This complexity leads to poor prognosis, which poses major challenges in the treatment of SLE. Understanding the complex genetic pathways and regulatory mechanisms operative in SLE was feasible by utilizing several highly efficient molecular biological tools during the past few years. In this perspective, DNA microarray technology offered a high-throughput platform in unraveling SLE-associated genes. Additionally, extensive microarray analysis had demonstrated aberrant DNA methylation pattern and differential microRNAs, thus contributing to the knowledge of epigenetic modulators and posttranscriptional regulatory machinery in SLE. It was through the aid of these technologies that interferon signature was identified as an important contributor in SLE pathogenesis along with dysregulation of cytokine-, chemokine- and apoptosis-related genes. The emergence of next-generation sequencing technologies such as RNA sequencing has added new dimensions in understanding the dynamics of the disease processes. Compared with microarrays, deep sequencing has provided higher resolution in gene expression measurement along with identification of different splicing events, noncoding RNAs and novel loci in SLE. The focus, therefore, has now been shifted toward the identification of novel gene loci and their isoforms, and their implication in disease pathogenesis. This advancement in the technology from microarray to deep sequencing has helped in deciphering the molecular pathways involved in pathogenesis of SLE and opens new avenues to develop novel treatment strategies for SLE.

A piggyBac-based reporter system for scalable in vitro and in vivo analysis of 3' untranslated region-mediated gene regulation.

Regulation of messenger ribonucleic acid (mRNA) subcellular localization, stability and translation is a central aspect of gene expression. Much of this control is mediated via recognition of mRNA 3′ untranslated regions (UTRs) by microRNAs (miRNAs) and RNA-binding proteins. The gold standard approach to assess the regulation imparted by a transcript's 3′ UTR is to fuse the UTR to a reporter coding sequence and assess the relative expression of this reporter as compared to a control. Yet, transient transfection approaches or the use of highly active viral promoter elements may overwhelm a cell's post-transcriptional regulatory machinery in this context. To circumvent this issue, we have developed and validated a novel, scalable piggyBac-based vector for analysis of 3′ UTR-mediated regulation in vitro and in vivo. The vector delivers three independent transcription units to the target genome—a selection cassette, a turboGFP control reporter and an experimental reporter expressed under the control of a 3′ UTR of interest. The pBUTR (piggyBac-based 3′ UnTranslated Region reporter) vector performs robustly as a siRNA/miRNA sensor, in established in vitro models of post-transcriptional regulation, and in both arrayed and pooled screening approaches. The vector is robustly expressed as a transgene during murine embryogenesis, highlighting its potential usefulness for revealing post-transcriptional regulation in an in vivo setting.

Regulatory elements involved in the post-transcriptional control of stage-specific gene expression in Trypanosoma cruzi: a review

Trypanosoma cruzi, a protozoan parasite that causes Chagas disease, exhibits unique mechanisms for gene expression such as constitutive polycistronic transcription of protein-coding genes, RNA editing and trans-splicing. In the absence of mechanism controlling transcription initiation, organized subsets of T. cruzi genes must be post-transcriptionally co-regulated in response to extracellular signals. The mechanisms that regulate stage-specific gene expression in this parasite have become much clearer through sequencing its whole genome as well as performing various proteomic and microarray analyses, which have demonstrated that at least half of the T. cruzi genes are differentially regulated during its life cycle. In this review, we attempt to highlight the recent advances in characterising cis and trans-acting elements in the T. cruzi genome that are involved in its post-transcriptional regulatory machinery.

Spermatids do it differently! Paip2a—the essential regulator of spermiogenesis?

Spermatids do it differently! Paip2a—the essential regulator of spermiogenesis?

Characterization of Heterologous Multigene Operons in Transgenic Chloroplasts. Transcription, Processing, and Translation

The first characterization of transcriptional, posttranscriptional, and translational processes of heterologous operons expressed via the tobacco (Nicotiana tabacum) chloroplast genome is reported here. Northern-blot analyses performed on chloroplast transgenic lines harboring seven different heterologous operons revealed that polycistronic mRNA was the predominant transcript produced. Despite the lack of processing of such polycistrons, large amounts of foreign protein accumulation was observed in these transgenic lines, indicating abundant translation of polycistrons. This is supported by polysome fractionation assays, which allowed detection of polycistronic RNA in lower fractions of the sucrose gradients. These results show that the chloroplast posttranscriptional machinery can indeed detect and translate multigenic sequences that are not of chloroplast origin. In contrast to native transcripts, processed and unprocessed heterologous polycistrons were stable, even in the absence of 3' untranslated regions (UTRs). Unlike native 5'UTRs, heterologous secondary structures or 5'UTRs showed efficient translational enhancement independent of cellular control. Abundant read-through transcripts were observed in the presence of chloroplast 3'UTRs but they were efficiently processed at introns present within the native operon. Heterologous genes regulated by the psbA (the photosystem II polypeptide D1) promoter, 5' and 3'UTRs have greater abundance of transcripts than the endogenous psbA gene because transgenes were integrated into the inverted repeat region. Addressing questions about polycistrons, and the sequences required for their processing and transcript stability, are essential in chloroplast metabolic engineering. Knowledge of such factors would enable engineering of foreign pathways independent of the chloroplast complex posttranscriptional regulatory machinery.

Derivation and function of small interfering RNAs and microRNAs

Small interfering RNA (siRNA) duplexes are generally produced by Dicer cleavage of double-stranded RNAs of frequently exogenous origin and can induce the cleavage and degradation of mRNAs bearing an identical sequence. In contrast, microRNAs (miRNAs) are encoded within the eukaryotic genome as short RNA hairpin structures. While these pre-miRNAs are also processed by Dicer, mature miRNAs appear to function primarily by inhibiting the translation of mRNAs bearing multiple, partially mismatched target sites. Nevertheless, recent data argue that the posttranscriptional regulatory machinery utilized by siRNAs and miRNAs is largely or entirely identical. In this review, I will discuss recent progress in unraveling the RNA processing pathway utilized for the biosynthesis of mature miRNAs and argue that this pathway offers at least three distinct entry points for the functional expression of artificial siRNAs in vertebrate cells. While each of these entry points offers distinct advantages and disadvantages, they all have the potential to induce the effective knock-down of specific genes either in cell culture or in experimental animals.

Hepatic responses to inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase: a comparison of atorvastatin and simvastatin

We have compared the cellular responses to simvastatin (Simva) and atorvastatin (Atorva), two potent HMG-CoA reductase inhibitors. The two drugs exhibited similar IC 50's for inhibition of either rat or human reductase, and single oral dosing in rats showed the compounds to be nearly equipotent at inhibiting hepatic cholesterol synthesis. Treatment of rats with Simva or Atorva in the feed for four days yielded comparable inductions of hepatic reductase activity and reductase protein. For example, 0.05% Simva induced reductase activity 27.3±9.1 fold and 0.05% Atorva induced activity 26.9±4.7 fold. This adaptive response was also studied in HepG2 cells, a human hepatoblastoma line, cultured for 24 h in delipidated serum and then for an additional 24 h with Simva or Atorva. Over a broad range (10 nM–10 μM), both drugs caused similar inductions of reductase activity, reductase protein, and reductase mRNA. Under all conditions, the drugs induced similar changes in the ratio of mRNA/protein suggesting that Simva and Atorva have similar effects on both transcriptional and post-transcriptional regulatory machinery. Moreover, reductase in cells treated with Simva or Atorva for 22 h responded similarly to subsequent challenge with 25-hydroxycholesterol. Finally, we measured the ability of the two reductase inhibitors to reduce ApoB secretion by HepG2 cells. Simva and Atorva at 0.5 μM inhibited ApoB secretion nearly identically, 38% and 42% respectively. We conclude that these two drugs induce similar adaptive responses in cells and that their actions are qualitatively and mechanistically identical. Human studies have shown that plasma is cleared of Atorva much more slowly than it is of Simva. The large pharmacokinetic difference in man, rather than some difference in mechanism, is the most likely explanation for the finding that the equipotent dose ratio for cholesterol lowering in humans of Simva to Atorva is about 2/1.