Primary RNA Transcript Research Articles

Molecular Investigation of Mitochondrial RNA19 Role in the Pathogenesis of MELAS Disease.

In mammalian mitochondria, the processing of primary RNA transcripts involves a coordinated series of cleavage and modification events, leading to the formation of processing intermediates and mature mt-RNAs. RNA19 is an unusually stable unprocessed precursor, physiologically polyadenylated, which includes the 16S mt-rRNA, the mt-tRNALeuUUR and the mt-ND1 mRNA. These peculiarities, together with the alteration of its steady-state levels in cellular models with defects in mitochondrial function, make RNA19 a potentially important molecule for the physiological regulation of mitochondrial molecular processes as well as for the pathogenesis of mitochondrial diseases. In this work, we quantitatively and qualitatively examined RNA19 in MELAS trans-mitochondrial cybrids carrying the mtDNA 3243A>G transition and displaying a profound mitochondrial translation defect. Through a combination of isokinetic sucrose gradient and RT-qPCR experiments, we found that RNA19 accumulated and co-sedimented with the mitoribosomal large subunit (mt-LSU) in mutant cells. Intriguingly, exogenous expression of the isolated LARS2 C-terminal domain (Cterm), which was shown to rescue defective translation in MELAS cybrids, decreased the levels of mt-LSU-associated RNA19 by relegating it to the pool of free unbound RNAs. Overall, the data reported here support a regulatory role for RNA19 in mitochondrial physiopathological processes, designating this RNA precursor as a possible molecular target in view of therapeutic strategy development.

The role of TDP-43 protein in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease where both upper and lower motoneurons are damaged. Even though the pathogenesis of ALS is unclear, the TDP-43 aggregations and non-nuclear localization may be crucial to understanding this process. Despite intensive research on ALS therapies, only two lifespan-prolonging medications have been approved: Riluzole and Edaravone. Unravelling the TDP-43 pathology could help develop new ALS therapies using mechanisms such as inhibition of nuclear export, autophagy, chaperones, or antisense oligonucleotides. Selective inhibitors of nuclear export (SINEs) are drugs that block Exportin 1 (XPO1) and cause the accumulation of not exported molecules inside the nucleus. SINEs that target XPO1 are shown to slightly extend the survival of neurons and soften motor symptoms. Dysfunctional proteins, including TDP-43, can be eliminated through autophagocytosis, which is regulated by the mTOR kinase. Stimulating the elimination of protein deposits may be an effective ALS therapy. Antisense oligonucleotides (ASO) are single-stranded, synthetic oligonucleotides that can bind and modulate specific RNA: via ribonuclease H, inducing their degradation or inducing alternative splicing via blocking primary RNA transcripts. Current ASOs therapies used in ALS focus on SOD1, C9ORF72, FUS, and ATXN2, and they may be used to slow the ALS progression. Reversing the aggregation is a promising therapeutic strategy. Chaperones control other proteins' quality and protect them against stress factors. Due to the irreversible character of ALS, it is essential to understand its complicated pathology better and to seek new therapies.

Retroviral RNA Processing.

This review is an accompaniment to a Special Issue on “Retroviral RNA Processing”. It discusses post-transcriptional regulation of retroviruses, ranging from the ancient foamy viruses to more modern viruses, such as HIV-1, HTLV-1, Rous sarcoma virus, murine leukemia virus, mouse mammary tumor virus, and Mason-Pfizer monkey virus. This review is not comprehensive. However, it tries to address some of the major questions in the field with examples of how different retroviruses express their genes. It is amazing that a single primary RNA transcript can have so many possible fates: genomic RNA, unspliced mRNA, and up to 50 different alternatively spliced mRNAs. This review will discuss the sorting of RNAs for packaging or translation, RNA nuclear export mechanisms, splicing, translation, RNA modifications, and avoidance of nonsense-mediated RNA decay.

ISG20 and nuclear exosome promote destabilization of nascent transcripts for spliceosomal U snRNAs and U1 variants

Primary RNA transcripts are processed in a plethora of ways to become mature functional forms. In one example, human spliceosomal U snRNAs are matured at their 3'-end by an exonuclease termed TOE1. This process is important because mutations in TOE1 gene can cause a human genetic disease, pontocerebellar hypoplasia (PCH). Nevertheless, TOE1 may not be the only maturation exonuclease for U snRNAs in the cell. Here, we biochemically identify two exonucleolytic factors, Interferon-stimulated gene 20-kDa protein (ISG20) and the nuclear exosome as such candidates, using a newly developed in vitro system that recapitulates 3'-end maturation of U1 snRNA. However, extensive 3'-end sequencing of endogenous U1 snRNA of the knockdown (KD) cells revealed that these factors are not the maturation factors per se. Instead, the nascent transcripts of the spliceosomal U snRNAs as well as of unstable U1 variants were found to increase in quantity upon KD of the factors. These results indicated that ISG20 and the nuclear exosome promote the degradation of nascent spliceosomal U snRNAs and U1 variants, and therefore implied their role in the quality control of newly synthesized U snRNAs.

Coordinated Mis-Splicing of Multiple Mitochondrial Iron Metabolism Genes Causes Ring Sideroblast Formation in SF3B1-Mutant MDS

Splicing is a fundamental process by which introns are removed from primary RNA transcripts. Alternative splicing is a major mechanism of gene regulation by which eukaryotic cells expand their transcriptional repertoire. By contrast, aberrant splicing generates novel transcripts not found in normal cells. Heterozygous gain-of-function mutations in a core U2 spliceosome factor SF3B1 are present in ~25% of myelodysplastic syndrome (MDS) patients. Strikingly, SF3B1 mutations are present in ~80% of MDS with ring sideroblasts (MDS-RS) patients suggesting a causal connection between mutant SF3B1 and ring sideroblasts (RS), erythroid precursors with iron-laden mitochondria (Yoshida et al. Nature 2011, Papaemmanuil et al.NEJM 2011). However, the mechanism by which SF3B1 mutations cause RS formation remains poorly understood since existing models of SF3B1-mutant MDS do not recapitulate RS formation in vitro. We have established an induced pluripotent stem cell (iPSC) model of MDS-RS that recapitulates mutant SF3B1-mediated mis-splicing and in vitro ring sideroblast formation. We reprogrammed SF3B1-mutant and SF3B1-wild-type iPSCs from individual MDS-RS patients enabling internal normalization to the isogenic normal clone (Hsu et al. Blood 2019). We established expandable multipotential HPC lines by conditional expression of five transcription factors (5F-HPC), followed by an 18-day erythroid differentiation. We monitored RS formation during the erythroid differentiation of 5F-HPCs using Prussian blue histological staining. RS formation increased during terminal erythropoiesis and peaked at 25-40% on day 18 in SF3B1-mutant lines, whereas SF3B1-wild-type lines showed no detectable RS formation. The frequency of RS was similar in isogenic SF3B1-only and SF3B1/EZH2 co-mutant cells suggesting that the mutant SF3B1 is sufficient to drive RS formation. To identify mis-splicing events that contribute to RS formation, we performed RNA-sequencing and splicing analysis of SF3B1-mutant and SF3B1-wild-type iPSCs at three stages of erythroid differentiation: CD34+ progenitor, CD71+ early erythroblast, and CD71+Glycophorin A+ erythroblast. Global isoform usage was dramatically altered during erythropoiesis, but was more similar in stage-matched SF3B1-mutant and SF3B1-wild-type cells suggesting that mutant SF3B1 selectively mis-splices a subset of transcripts. We identified 2300 transcripts with >10% mis-splicing in SF3B1-mutant lines, and only 120 transcripts with more significant >40% mis-splicing. Of these, TMEM14C and PPOX, inner mitochondrial membrane components of the heme synthesis pathway were strongly mis-spliced throughout erythroid differentiation, consistent with previous studies (Conte et al.BJHaem 2015, Shiozawa et al. Nat Commun 2018). The transcript levels of PPOX but not TMEM14C were reduced in SF3B1-mutant lines as a result of mis-splicing. The expression of ABCB7, a mitochondrial iron sulfur cluster biogenesis component mutated in inherited X-linked sideroblastic anemia (Allikmets et al. Hum Mol Genet 1999), was also reduced in SF3B1-mutant cells as expected (Shiozawa et al. Nat Commun 2018, Dolatshad et al. Leukemia 2016). To investigate the role of these mis-splicing events in ring sideroblast formation, we performed lentiviral overexpression of TMEM14C, PPOX, and ABCB7, in SF3B1-mutant 5F-HPCs and quantified RS formation during late stages of erythroid differentiation. Overexpression of TMEM14C and ABCB7 in SF3B1-mutant cells partially but not completely rescued RS formation compared to luciferase control. These findings confirm the long-standing hypothesis that mis-splicing of mitochondrial iron metabolism genes causes RS formation. Furthermore, these findings suggest that RS formation in MDS is a multigenic event caused by coordinated but incomplete mis-splicing of several critical iron metabolism genes. Disclosures No relevant conflicts of interest to declare.

Overview on Keloid Disorder: Phenotypic Spectrum, Connective Tissue Pathology, and Treatment Development

Overview on Keloid Disorder: Phenotypic Spectrum, Connective Tissue Pathology, and Treatment Development

Maheshvara, a Conserved RNA Helicase, Regulates Notch Signaling in Drosophila melanogaster.

Gene expression is regulated at multiple steps after generation of primary RNA transcripts, including mRNA processing, stability, and transport, along with co- and post-transcriptional regulation. These processes are controlled via the involvement of a multitude of RNA binding proteins (RBPs). Innumerable human diseases have been associated with altered expression of RNA binding proteins. In this chapter we have focused on Maheshvara (mahe) which encodes a putative DEAD box RNA helicase protein in Drosophila. We have recently reported that mahe plays an important role in regulation of Notch signaling. Fine tuning of Notch signaling is required at multiple stepsand it's misregulation leads to a variety of human diseases. Additionally, mutation in DDX59, a human homolog of mahe results in broad neurological phenotypesassociated with orofaciodigital syndrome. Drosophila mahe mutants show abnormal peripheral and central nervous system development that resemble neuropathology of patients having mutation in DDX59 gene. This chapter will help in advancing the knowledge as to how mahe regulates Notch signaling and nervous system development.

Probing Aberrant Splicing in a Novel Model of SF3B1-Mutant Myelodysplastic Syndromes

Splicing is a fundamental process by which introns are removed from primary RNA transcripts. Alternative splicing is widely appreciated to be a major mechanism of gene regulation by which eukaryotic cells expand their transcriptional repertoire. By contrast, aberrant splicing generates novel transcripts not found in normal cells. Heterozygous mutations in a core U2 spliceosome factor SF3B1 are strongly associated with MDS with ring sideroblasts (MDS-RS). MDS-RS is characterized by iron retention in mitochondria and ineffective erythropoiesis. How aberrant splicing induced by mutant SF3B1 causes ring sideroblasts and erythroid defects remains poorly understood. There are no genetically accurate models of MDS-RS that recapitulate ring sideroblast formation. Our lab has established an iPSC reprogramming approach to generate an in vitro model of MDS-RS. This method captures genetically distinct MDS subclones as well as normal cells in individual patients enabling internal normalization to the patient's isogenic normal clone. By differentiating iPSCs we generated inducibly immortalized MDS-RS CD34+ progenitor cell lines with robust expansion and erythroid differentiation potential enabling extensive analysis of SF3B1-mutant aberrant splicing throughout erythroid differentiation. We show that SF3B1-mutant lines form abundant ring sideroblasts compared to normal isogenic controls. We next performed RNA-sequencing and splicing analysis at different stages of erythroid differentiation. We show that global splicing patterns are maintained in SF3B1-mutant cells, while revealing distinct subsets of aberrantly spliced transcripts in CD34+ progenitors and erythroblast populations. This suggests that different aberrant splicing events contribute to erythroid progenitor expansion and inefficient erythropoiesis and iron dysregulation in MDS-RS. Taken together, this novel iPSC model of MDS-RS is a robust tool for studying the role of aberrant splicing in SF3B1-mutant erythroid progenitor expansion, inefficient erythropoiesis and ring sideroblast formation. Disclosures No relevant conflicts of interest to declare.

DeepDSSR: Deep Learning Structure for Human Donor Splice Sites Recognition.

Human genes often, through alternative splicing of pre-messenger RNAs, produce multiple mRNAs and protein isoforms that may have similar or completely different functions. Identification of splice sites is, therefore, crucial to understand the gene structure and variants of mRNA and protein isoforms produced by the primary RNA transcripts. Although many computational methods have been developed to detect the splice sites in humans, this is still substantially a challenging problem and further improvement of the computational model is still foreseeable. Accordingly, we developed DeepDSSR (deep donor splice site recognizer), a novel deep learning based architecture, for predicting human donor splice sites. The proposed method, built upon publicly available and highly imbalanced benchmark dataset, is comparable with the leading deep learning based methods for detecting human donor splice sites. Performance evaluation metrics show that DeepDSSR outperformed the existing deep learning based methods. Future work will improve the predictive capabilities of our model, and we will build a model for the prediction of acceptor splice sites.

Interpreting the Dynamics of Binding Interactions of snRNA and U1A Using a Coarse-Grained Model

The binding interactions of small nuclear RNAs (snRNA) and the associated protein factors are critical to the function of spliceosomes in alternatively splicing primary RNA transcripts. Although molecular dynamics simulations are a powerful tool to interpret the mechanism of biological processes, the atomic-level simulations are, however, too expensive and with limited accuracy for the large-size systems, such as snRNA-protein complexes. We extend the coarse-grained Gaussian network model, which models the RNA-protein complexes as a harmonic chain of Cα, P, and O4′ atoms, to investigating the impact of the snRNA-binding interaction on the dynamic stability of the human U1A protein, which is a major component of the spliceosomal U1 small nuclear ribonucleoprotein particle. The results reveal that the first and third loops and the C-terminal helix regions of the U1A domain undergo a significant loss of flexibility upon the RNA binding due to the forming of mostly electrostatic and hydrogen bond interactions with RNA 5′ stem and loop. By examining the residues whose mutations significantly change the binding free energy between U1A and snRNA, the Gaussian network model-based calculations show that not only the residues at the binding sites that are traditionally considered to play a major role in U1A-RNA association but also those residues that are far away from the RNA-binding interface can participate in the long-range allosteric signal transmission; these calculations are quantitatively consistent with the data observed in the recent snRNA binding experiments. The study demonstrates a useful avenue to utilize the simplified elastic network model to investigate the dynamics characteristics of the biologically important macromolecular interactions.

StarSeeker: an automated tool for mature duplex microRNA sequence identification based on secondary structure modeling of precursor molecule

BackgroundMicroRNAs (miRNAs) are small, non-coding RNA molecules that play a key role in gene regulation in both plants and animals. MicroRNA biogenesis involves the enzymatic processing of a primary RNA transcript. The final step is the production of a duplex molecule, often designated as miRNA:miRNA*, that will yield a functional miRNA by separation of the two strands. This miRNA will be incorporated into the RNA-induced silencing complex, which subsequently will bind to its target mRNA in order to suppress its expression. The analysis of miRNAs is still a developing area for computational biology with many open questions regarding the structure and function of this important class of molecules. Here, we present StarSeeker, a simple tool that outputs the putative miRNA* sequence given the precursor and the mature sequences.ResultsWe evaluated StarSeeker using a dataset consisting of all plant sequences available in miRBase (6992 precursor sequences and 8496 mature sequences). The program returned a total of 15,468 predicted miRNA* sequences. Of these, 2650 sequences were matched to annotated miRNAs (~ 90% of the miRBase-annotated sequences). The remaining predictions could not be verified, mainly because they do not comply with the rule requiring the two overhanging nucleotides in the duplex molecule.ConclusionsThe expression pattern of some miRNAs in plants can be altered under various abiotic stress conditions. Potential miRNA* molecules that do not degrade can thus be detected and also discovered in high-throughput sequencing data, helping us to understand their role in gene regulation.

MIR205HG is a Long Noncoding RNA with Distinct Functions in the Thymus versus the Anterior Pituitary

Abstract MicroRNAs (miRNAs) are processed from primary RNA transcripts (pri-miRNAs) encoded in host genes. Several miRNAs including miR-205 are present within long noncoding RNAs (lncRNAs). MiR-205 is an epithelial-specific miRNA that supports thymopoiesis by positively regulating Forkhead Box N1 (Foxn1) expression. We assessed whether the host gene for miR-205, MIR205HG, has independent functions as a lncRNA. The more severe stress-induced thymic atrophy reported in miR-205-deficient mice is also evident in MIR205HG knockout lines. However, MIR205HG knockouts have a small stature phenotype. A new set of miR-205KI mice did not have this phenotype. The smaller mouse size of the MIR205HG null animals is partly a consequence of reduced levels of endocrine hormones produced by the anterior pituitary. In addition, the MIR205HG null animals had abnormal development of the lacrimal and harderian glands that produce eye secretions. Transcriptome analyses revealed that MIR205HG regulates gene expression in the anterior pituitary unlike miR-205. In contrast, transcripts regulated by miR-205 and MIR205HG in the epithelial cells of the skin and thymus overlap significantly. These data indicate that MIR205HG has a specific function as a lncRNA in the anterior pituitary in addition to its primary role as the host gene for miR-205 in thymic epithelial cells.

RNA-binding proteins implicated in neurodegenerative diseases.

Gene expression is regulated at many levels, including after generation of the primary RNA transcript from DNA but before translation into protein. Such post-translational gene regulation occurs via the action of a multitude of RNA binding proteins and include varied actions from splicing to regulation of association with the translational machinery. Primary evidence that such processes might contribute to disease mechanisms in neurodegenerative disorders comes from the observation of mutations in RNA binding proteins, particularly in diseases in the amyotrophic lateral sclerosis-frontotemporal dementia spectrum and in some forms of ataxia and tremor. The bulk of evidence from recent surveys of the types of RNA species that are affected in these disorders suggests a global deregulation of control rather than a very small number of RNA species, although why some groups of neurons are sensitive to these changes is not well understood. Overall, these data suggest that neurodegeneration can be initiated by mutations in RNA binding proteins and, as a corollary, that neurons are particularly sensitive to loss of control of gene expression at the post-transcriptional level. Such observations have implications not only for understanding the nature of neurodegenerative disorders but also how we might intervene therapeutically in these diseases. WIREs RNA 2017, 8:e1397. doi: 10.1002/wrna.1397 For further resources related to this article, please visit the WIREs website.

A reliable method for quantification of splice variants using RT-qPCR.

BackgroundThe majority of protein isoforms arise from alternative splicing of the encoding primary RNA transcripts. To understand the significance of single splicing events, reliable techniques are needed to determine their incidence. However, existing methods are labour-intensive, error-prone or of limited use.ResultsHere, we present an improved method to determine the relative incidence of transcripts that arise from alternative splicing at a single site. Splice variants were quantified within a single sample using one-step reverse transcription quantitative PCR. Amplification products obtained with variant specific primer pairs were compared to those obtained with primer pairs common to both variants. The identities of variant specific amplicons were simultaneously verified by melt curve analysis. Independent calculations of the relative incidence of each variant were performed. Since the relative incidences of variants have to add upto 100 %, the method provides an internal control to monitor experimental errors and uniform reverse transcription. The reliability of the method was tested using mixtures of cDNA templates as well as RNA samples from different sources.ConclusionThe method described here, is easy to set up and does not need unrelated reference genes and time consuming, error-prone standard curves. It provides a reliable and precise technique to distinguish small differences of the relative incidence of two splice variants.

The Drosophila Helicase MLE Targets Hairpin Structures in Genomic Transcripts.

RNA hairpins are a common type of secondary structures that play a role in every aspect of RNA biochemistry including RNA editing, mRNA stability, localization and translation of transcripts, and in the activation of the RNA interference (RNAi) and microRNA (miRNA) pathways. Participation in these functions often requires restructuring the RNA molecules by the association of single-strand (ss) RNA-binding proteins or by the action of helicases. The Drosophila MLE helicase has long been identified as a member of the MSL complex responsible for dosage compensation. The complex includes one of two long non-coding RNAs and MLE was shown to remodel the roX RNA hairpin structures in order to initiate assembly of the complex. Here we report that this function of MLE may apply to the hairpins present in the primary RNA transcripts that generate the small molecules responsible for RNA interference. Using stocks from the Transgenic RNAi Project and the Vienna Drosophila Research Center, we show that MLE specifically targets hairpin RNAs at their site of transcription. The association of MLE at these sites is independent of sequence and chromosome location. We use two functional assays to test the biological relevance of this association and determine that MLE participates in the RNAi pathway.

Regulation of IgD Expression and Its Role in B Cell Transformation

A developing B lymphocyte first acquires expression of immunoglobulin M (IgM) at the immature stage, and along maturation into the transitional stage it starts to co-express another isotype-IgD. IgM and IgD are generated by alternative splicing of long primary RNA transcripts from the Ig heavy chain (Igh) locus, so that they share the N-terminal antigen-binding variable region, but differ in the C-terminal constant region, encoded by Cμ or Cδ exons, respectively. Maturation into follicular (naïve) B cells, IgD levels surpass those of IgM; while upon antigen-stimulation and entering germinal center reaction, follicular B cells turn off IgD expression. Trans-acting factors have been proposed to control IgM and IgD expression since several decades ago. However, the first of such factors, Zfp318, was just reported last year (Enders et al. Proc Natl Acad Sci USA 2014; Pioli et al. J Immunol 2014). Still, other factors and signaling cascades regulating IgD expression remain to be identified.In this context, we accidentally discovered that expression of the Epstein-Barr virus (EBV) oncoprotein latent membrane protein 1 (LMP1) in B cells specifically abolishes IgD but not IgM expression; LMP1 signaling suppresses IgD expression at the mRNA level. Using microarray we screened differentially expressed genes in LMP1-expressing versus normal mouse B cells, and then looked up these genes in ImmuGen database (http://www.immgen.org) to identify those whose expression patterns correlate with that of IgD at various B cell developmental stages. These analyses returned us two new candidates Rapgef4 and CD55, in addition to Zfp318. By CRISPR technology, we further demonstrated that mutation of Rapgef4 or CD55 in mature B cells results in downregulation of IgD but not IgM. To define their role in regulating IgD expression in vivo, mice carrying conditional null alleles Rapgef4F/F and CD55F/F, are being generated.The facts that IgD is an evolutionarily conserved Ig isotype and its expression is precisely regulated during B cell developmental and functional stages suggest an important role in B cell biology/function. Indeed, a recent study suggested that IgD, by sequestering CD19 coreceptor from IgM, might play a negative role in B cell receptor (BCR) signaling (Klasener et al. eLife 2014). Interestingly, flow cytometric analysis of 12 diffuse large B cell lymphoma (DLBCL)-like malignancies arising in our mouse model revealed that 10 cases are low/negative for IgD and the other 2 cases express intermediate levels of IgD, while they all express surface IgM or IgG (Zhang et al. Cell Rep 2015; data not shown); human DLBCLs often retain surface IgM expression (Wright et al. Proc Natl Acad Sci USA 2003; IgD expression data not available). Basing on aforementioned observations, we hypothesize that downregulation of IgD may be important for LMP1-induced B cell transformation and for DLBCL pathogenesis in general. Accordingly, in this work, we will reconstitute IgD expression in LMP1+ lymphoma cell lines (Zhang et al. Cell 2012), and determine if it suppresses lymphoma cell growth in vitro and in vivo. Similarly, we will reconstitute IgD expression in a few IgD- mouse DLBCL lines and monitor its impact on lymphoma growth. In addition, as we have 2 DLBCL lines expressing intermediate levels of IgD, we will use CRISPR technology to delete/mutate Cδ coding sequence in these lymphoma lines, and see if the mutated cells (losing IgD expression) outcompete the non-mutated cells.Overall, this work will lead to molecular insights into IgD regulation. It may also elucidate the functional significance of IgD downregulation in B cell lymphoma pathogenesis/maintenance, and guide therapy development targeting BCR signaling. DisclosuresNo relevant conflicts of interest to declare.

A-to-I RNA Editing: Current Knowledge Sources and Computational Approaches with Special Emphasis on Non-Coding RNA Molecules

RNA editing is a dynamic mechanism for gene regulation attained through the alteration of the sequence of primary RNA transcripts. A-to-I (adenosine-to-inosine) RNA editing, which is catalyzed by members of the adenosine deaminase acting on RNA (ADAR) family of enzymes, is the most common post-transcriptional modification in humans. The ADARs bind double-stranded regions and deaminate adenosine (A) into inosine (I), which in turn is interpreted by the translation and splicing machineries as guanosine (G). In recent years, this modification has been discovered to occur not only in coding RNAs but also in non-coding RNAs (ncRNA), such as microRNAs, small interfering RNAs, transfer RNAs, and long non-coding RNAs. This may have several consequences, such as the creation or disruption of microRNA/mRNA binding sites, and thus affect the biogenesis, stability, and target recognition properties of ncRNAs. The malfunction of the editing machinery is not surprisingly associated with various human diseases, such as neurodegenerative, cardiovascular, and carcinogenic diseases. Despite the enormous efforts made so far, the real biological function of this phenomenon, as well as the features of the ADAR substrate, in particular in non-coding RNAs, has still not been fully understood. In this work, we focus on the current knowledge of RNA editing on ncRNA molecules and provide a few examples of computational approaches to elucidate its biological function.

Microrna-106b~25 Cluster Is Involved in Relapsed MLL-Rearranged Pediatric AML

AbstractMixed lineage leukemia (MLL) rearrangements represent about 20% of pediatric acute myeloid leukemia (AML) cases. Survival rates have increased over the past decades due to intensified chemotherapy protocols and improved supportive care. Still, approximately 30-40% will relapse during or after therapy.The role of microRNAs (miRNAs) in leukemogenesis of MLL-rearranged AML, especially towards the development of relapse is unknown. To determine whether specific miRNAs are involved in relapse development, we performed miRNA profiling, using TaqMan Low Density Array (TLDA) in pediatric AML with a special focus on MLL-rearranged cases. First, we compared miRNA profiles of de novo pediatric AML patients that relapsed with those that did not relapse. Secondly, we did the same comparison, only focussing on the MLL-rearranged subset. Third, we investigated the role of miRNAs in clonal evolution by investigating the differential expression of 6 paired initial diagnosis-relapses MLL-rearranged AML cases. An independent set of 6 paired initial diagnosis-relapse cases with MLL-rearrangements was used to confirm the identified miRNAs using single stemloop RT-qPCR.In a cohort of 127 de novo AML cases, 59/127 (46.5%) relapsed after complete remission. Comparing miRNA profiles of relapsing and non-relapsing cases, we could not identify differentially expressed miRNAs signatures between the two groups at diagnosis (p=0.643). Likewise, in a nested cohort of de novo pediatric AML MLL-rearranged cases (4 AF6, 7 AF9, 9 AF10, 1 FNBP1, 1 SEPT6, and 1 NRIP3), of which 14/23 (60.9%) relapsed we did not identify differentially expressed microRNAs (p=0.429).In the 6 paired initial diagnosis-relapse MLL-rearranged cases (2 AF9 and 4 AF10), 53 miRNAs were significantly differentially expressed (FDR <0.1).Among those, miR-106b, miR-93 and miR-25, were most highly overexpressed at relapse. These microRNAs cluster together in intron 13 of minichromosome maintenance complex component 7 (MCM7) and are actively cotranscribed in the context of MCM7 primary RNA transcript. E2F transcription factor 1 (E2F1) acts as an upstream regulator. Overexpression of MCM7has been associated with poor prognosis in solid cancers and this may be linked to overexpression of the hosted microRNAs, the miR-106b~25 cluster. A possible role of the miR-106b~25 cluster in leukemia has so far not been investigated.Therefore, we validated the expression of MCM7 and E2F1 mRNA (RT-qPCR) and found them to be differentially overexpressed in the paired samples with MLL-rearrangements at relapse (n=12, p=0.006 and p=0.003, respectively). Overexpression of miR-106b and miR-25 at relapse was confirmed by stem loop RT-qPCR in the patient samples as well as in 6 additional paired samples with MLL-rearrangements at relapse (4 AF10, 1 ELL, and 1 unknown)(n=12, p<0.05). MiR-93 overexpression could however not be confirmed by stem loop RT-qPCR (p=0.08).Expression levels of two other predicted targets of the miR-106b~25 cluster as taken from the literature (Petrocca et al., 2008), cyclin-dependent kinase inhibitor 1A (p21WAF1/CIP1) and BCL2-like 11 (BIM), were analysed in 12 paired initial diagnosis-relapse samples. We did not find differences in mRNA expression of p21WAF1/CIP1 (p=0.17) or BIM (p=0.27) between initial diagnosis and relapse. As microRNAs regulate genes expression through translational repression or target mRNA cleavage we investigated protein level (Western blot, n=3 paired samples) of p21WAF1/CIP1, BIM, MCM7, and E2F1. In case of mRNA cleavage, a reverse correlation between predicted target gene and miRNA is expected. In 2/3 paired initial diagnosis-relapse samples the expression of p21WAF1/CIP1, BIM, E2F1, and MCM7 was downregulated in relapse samples as compared to initial diagnosis. Only one patient showed increased protein expression of E2F1 and MCM7, a modest downregulation of BIM, and no expression of p21WAF1/CIP1.Together, our data indicate that miR-106b-25 cluster may play an important role in relapse pediatric AML with MLL-rearrangements. Further research is warranted to identify the role of this cluster for clinical resistance and as treatment target. DisclosuresNo relevant conflicts of interest to declare.

Modulation of proopiomelanocortin gene expression by ethanol in mouse anterior pituitary corticotrope tumor cell AtT20

In humans, alcoholism is associated with a decrease in basal ACTH and cortisol levels, and blunted pituitary ACTH responses to administered corticotropin-releasing hormone (CRH) during active drinking and after long-term abstinence. Preclinical studies indicate that a persistent decrease in pituitary activation after chronic exposure to ethanol is due to a direct effect of ethanol on the corticotrope of the anterior pituitary. The present studies were undertaken to determine if ethanol has effects on proopiomelanocortin (POMC) gene transcription activity in mouse anterior pituitary corticotrope tumor cell AtT20. We measured the levels of the POMC primary nuclear RNA transcript (PT), processing intermediate, and mature mRNA in the nucleus and the levels of the POMC mRNA in the cytoplasm after treatment of AtT20 cells with 5–15mM concentrations of ethanol. After 15mM ethanol for 60 to 120min, the POMC PT levels were significantly decreased. This decreased POMC gene transcription activity was coupled with a significant reduction of the POMC cytoplasmic mRNA levels. After ethanol for 4h, however, both the decreases were no longer observed. After 8h, a decrease in the ACTH secretion in the medium was found. We further investigated if CRH or glutamate modulates the effects of ethanol on the POMC gene transcription activity. CRH at 10nM after 60min increased the POMC PT levels, and 15mM ethanol attenuated the effect of CRH on the nuclear transcription activity. Glutamate receptor proteins, including NMDA receptor subtype NR1 (but not NR2A or NR2B) and GluR2, were identified by Western immunoblot analysis in AtT20 cells, with similar sizes to those in mouse hypothalamus. The inhibitory effect of 60min ethanol at 5 to 15mM on the POMC PT levels was attenuated by 50μML-glutamate. Together, our data showed that: (1) ethanol treatment in intoxicate doses significantly inhibited POMC gene transcription activity in a dose- and time-dependent manner in AtT20 cells, and (2) the POMC gene transcription activity in response to CRH or glutamate was altered by ethanol. Our results suggest that ethanol has an inhibitory effect on the POMC gene transcription activity in the anterior pituitary corticotrope, which may contribute to the persistent decrease in pituitary activation after chronic ethanol exposure.

Drosha promotes splicing of a pre-microRNA-like alternative exon.

The ribonuclease III enzyme Drosha has a central role in the biogenesis of microRNA (miRNA) by binding and cleaving hairpin structures in primary RNA transcripts into precursor miRNAs (pre-miRNAs). Many miRNA genes are located within protein-coding host genes and cleaved by Drosha in a manner that is coincident with splicing of introns by the spliceosome. The close proximity of splicing and pre-miRNA biogenesis suggests a potential for co-regulation of miRNA and host gene expression, though this relationship is not completely understood. Here, we describe a cleavage-independent role for Drosha in the splicing of an exon that has a predicted hairpin structure resembling a Drosha substrate. We find that Drosha can cleave the alternatively spliced exon 5 of the eIF4H gene into a pre-miRNA both in vitro and in cells. However, the primary role of Drosha in eIF4H gene expression is to promote the splicing of exon 5. Drosha binds to the exon and enhances splicing in a manner that depends on RNA structure but not on cleavage by Drosha. We conclude that Drosha can function like a splicing enhancer and promote exon inclusion. Our results reveal a new mechanism of alternative splicing regulation involving a cleavage-independent role for Drosha in splicing.