Changes In RNA Processing Research Articles

Breastfeeding reduces the risk of breast cancer: A call for action in high-income countries with low rates of breastfeeding.

Women in the UK have a 15% lifetime risk of developing breast cancer. Like other high-income countries, women in the UK are having children later in life which increases their risk. The risk of breast cancer is reduced by 4.3% for every 12 months of breastfeeding, this is in addition to the 7.0% decrease in risk observed for each birth. Breastfeeding reduces the risk of Triple-Negative Breast Cancer (20%) and in carriers of BRCA1 mutations (22-55%). The mechanisms of reduced risk as a result of pregnancy are related to changes in RNA processing and cellular differentiation. The UK has a low rate of breastfeeding (81%) and this is contrasted to countries with higher (Sweden, Australia) and lower rates (Ireland). The low UK rate is in part due to a lack of experience in the population, todays grandmothers have less experience with breastfeeding (62%) than their daughters. An estimated 4.7% of breast cancer cases in the UK are caused by not breastfeeding. The UK only has 43% of maternity services with full Baby-Friendly accreditation which promotes compliance with the WHO 'Ten Steps to Successful Breast Feeding'. Legislation in the UK and Europe is far short of the WHO Guidance on restricting the advertising of formula milk. Expansion of the Baby-Friendly Hospital Initiative, stricter laws on the advertising of formula milk and legislation to support nursing mothers in the workplace have the potential to increase breastfeeding in the UK. Women with a family history of breast cancer should particularly be supported to breastfeed as a way of reducing their risk.

Multiple functions of phospholipase Cβ1 at a glance.

Phospholipase Cβ (PLCβ) is the main effector of the Gq family of heterotrimeric G proteins that transduces signals from hormones and neurotransmitters into Ca2+ signals. While PLCβ is critical for Ca2+ responses, recent studies have suggested that PLCβ has additional roles independent of its lipase activity. These novel functions are carried out by a cytosolic population of PLCβ that binds and inhibits the component 3 promoter of RNA-induced silencing complex (C3PO) to impact cytosolic RNA populations. Additionally, cytosolic PLCβ binds to stress granule proteins, keeping them dispersed and thus inhibiting stress granule formation. Upon activation of the Gα subunit of Gq (Gαq), cytosolic PLCβ relocalizes to the membrane, releasing C3PO and stress granule proteins, which in turn promotes activation of C3PO and RNA processing, as well as sequestration of specific transcripts into newly formed stress granules. As highlighted in this Cell Science at a Glance and the accompanying poster, the link between Gαq signaling, increased intracellular Ca2+ and changes in RNA processing impacts neuronal cell differentiation and may also affect neuronal development and dysfunction.

The role of the RNA binding protein HnRNP K in the pathogenesis of frontotemporal lobar degeneration.

HnRNP K is an RNA-binding protein belonging to the family of heterogeneous nuclear ribonucleoproteins (hnRNPs). Like other hnRNPs such as the better known TDP-43, hnRNP K is predominantly nuclear where it plays an important role in pre-mRNA splicing. HnRNPs can also shuttle to the cytoplasm and we find that hnRNP K is predominantly cytoplasmic in pyramidal neurons in Frontotemporal Lobar Degeneration (FTLD). The loss of nuclear hnRNPs in disease states is directly linked to splicing misregulation and aberrant RNA processing. Indeed, loss of nuclear TDP-43 leads to cryptic exon inclusion in gene targets and the generation of truncated proteins. We aimed to investigate changes in RNA processing and presence of cryptic events upon hnRNP K mislocalisation in FTLD. Bioinformatic analysis of a neuronal cell line with hnRNP K knockdown identified a set of cryptic events. We have validated these events using PCR amplification and gel electrophoresis in both SH-SY5Y cells with hnRNP K knockdown and in post-mortem human brain tissue. The loss of nuclear hnRNP K results in the inclusion of cryptic exons in multiple gene targets in both SH-SHY5Y cells with hnRNP K knockdown and FTLD brain regions exhibiting hnRNP K nuclear loss. The mislocalisation of hnRNP K from the nucleus to the cytoplasm in FTLD is associated with the inclusion of cryptic exons in multiple gene targets. This suggests that similarly to TDP-43, hnRNP K plays a crucial role in RNA processing and splicing regulation which when impaired could lead to transcriptional dysregulation and disease pathogenesis.

A CRISPR RNA-binding protein screen reveals regulators of RUNX1 isoform generation

AbstractThe proper balance of hematopoietic stem cell (HSC) self-renewal and differentiation is critical for normal hematopoiesis and is disrupted in hematologic malignancy. Among regulators of HSC fate, transcription factors have a well-defined central role, and mutations promote malignant transformation. More recently, studies have illuminated the importance of posttranscriptional regulation by RNA-binding proteins (RBPs) in hematopoiesis and leukemia development. However, the RBPs involved and the breadth of regulation are only beginning to be elucidated. Furthermore, the intersection between posttranscriptional regulation and hematopoietic transcription factor function is poorly understood. Here, we studied the posttranscriptional regulation of RUNX1, a key hematopoietic transcription factor. Alternative polyadenylation (APA) of RUNX1 produces functionally antagonistic protein isoforms (RUNX1a vs RUNX1b/c) that mediate HSC self-renewal vs differentiation, an RNA-processing event that is dysregulated in malignancy. Consequently, RBPs that regulate this event directly contribute to healthy and aberrant hematopoiesis. We modeled RUNX1 APA using a split GFP minigene reporter and confirmed the sensitivity of our model to detect changes in RNA processing. We used this reporter in a clustered regularly interspaced short palindromic repeats (CRISPR) screen consisting of single guide RNAs exclusively targeting RBPs and uncovered HNRNPA1 and KHDRBS1 as antagonistic regulators of RUNX1a isoform generation. Overall, our study provides mechanistic insight into the posttranscriptional regulation of a key hematopoietic transcription factor and identifies RBPs that may have widespread and important functions in hematopoiesis.

Molecular and phenotypic analysis of rodent models reveals conserved and species-specific modulators of human sarcopenia

Sarcopenia, the age-related loss of skeletal muscle mass and function, affects 5–13% of individuals aged over 60 years. While rodents are widely-used model organisms, which aspects of sarcopenia are recapitulated in different animal models is unknown. Here we generated a time series of phenotypic measurements and RNA sequencing data in mouse gastrocnemius muscle and analyzed them alongside analogous data from rats and humans. We found that rodents recapitulate mitochondrial changes observed in human sarcopenia, while inflammatory responses are conserved at pathway but not gene level. Perturbations in the extracellular matrix are shared by rats, while mice recapitulate changes in RNA processing and autophagy. We inferred transcription regulators of early and late transcriptome changes, which could be targeted therapeutically. Our study demonstrates that phenotypic measurements, such as muscle mass, are better indicators of muscle health than chronological age and should be considered when analyzing aging-related molecular data.

Noncanonical functions of the serine-arginine-rich splicing factor (SR) family of proteins in development and disease.

Members of the serine/arginine (SR)-rich protein family of splicing factors play versatile roles in RNA processing steps and are often essential for normal development. Dynamic changes in RNA processing and turnover allow fast cellular adaptions to a changing microenvironment and thereby closely cooperate with transcription factor networks that establish cell identity within tissues. SR proteins play fundamental roles in the processing of pre-mRNAs by regulating constitutive and alternative splicing. More recently, SR proteins have also been implicated in other aspects of RNA metabolism such as mRNA stability, transport and translation. The- emerging noncanonical functions highlight the multifaceted functions of these SR proteins and identify them as important coordinators of gene expression programmes. Accordingly, most SR proteins are essential for normal cell function and their misregulation contributes to human diseases such as cancer.

Gene co-expression analysis reveals transcriptome divergence between wild and cultivated chickpea under drought stress.

Ancestral adaptations in crop wild relatives can provide a genetic reservoir for crop improvement. Here we document physiological changes to mild and severe drought stress, and the associated transcriptome dynamics in both wild and cultivated chickpea. Over 60% of transcriptional changes were related to metabolism, indicating that metabolic plasticity is a core and conserved drought response. In addition, changes in RNA processing and protein turnover were predominant in the data, suggestive of broad restructuring of the chickpea proteome in response to drought. While 12% of the drought-responsive transcripts have similar dynamics in cultivated and wild accessions, numerous transcripts had expression patterns unique to particular genotypes, or that distinguished wild from cultivated genotypes and whose divergence may be a consequence of domestication. These and other comparisons provide a transcriptional correlate of previously described species' genetic diversity, with wild accessions well differentiated from each other and from cultivars, and cultivars essentially indistinguishable at the broad transcriptome level. We identified metabolic pathways such as phenylpropanoid metabolism, and biological processes such as stomatal development, which are differentially regulated across genotypes with potential consequences on drought tolerance. These data indicate that wild Cicer reticulatum may provide both conserved and divergent mechanisms as a resource in breeding for drought tolerance in cultivated chickpea.

Transcriptome alterations in myotonic dystrophy skeletal muscle and heart.

Myotonic dystrophy (dystrophia myotonica, DM) is a multi-systemic disease caused by expanded CTG or CCTG microsatellite repeats. Characterized by symptoms in muscle, heart and central nervous system, among others, it is one of the most variable diseases known. A major pathogenic event in DM is the sequestration of muscleblind-like proteins by CUG or CCUG repeat-containing RNAs transcribed from expanded repeats, and differences in the extent of MBNL sequestration dependent on repeat length and expression level may account for some portion of the variability. However, many other cellular pathways are reported to be perturbed in DM, and the severity of specific disease symptoms varies among individuals. To help understand this variability and facilitate research into DM, we generated 120 RNASeq transcriptomes from skeletal and heart muscle derived from healthy and DM1 biopsies and autopsies. A limited number of DM2 and Duchenne muscular dystrophy samples were also sequenced. We analyzed splicing and gene expression, identified tissue-specific changes in RNA processing and uncovered transcriptome changes strongly correlating with muscle strength. We created a web resource at http://DMseq.org that hosts raw and processed transcriptome data and provides a lightweight, responsive interface that enables browsing of processed data across the genome.

Identification and Characterization of Transcripts Regulated by Circadian Alternative Polyadenylation in Mouse Liver.

Dynamic control of gene expression is a hallmark of the circadian system. In mouse liver, approximately 5–20% of RNAs are expressed rhythmically, and over 50% of mouse genes are rhythmically expressed in at least one tissue. Recent genome-wide analyses unveiled that, in addition to rhythmic transcription, various post-transcriptional mechanisms play crucial roles in driving rhythmic gene expression. Alternative polyadenylation (APA) is an emerging post-transcriptional mechanism that changes the 3′-ends of transcripts by alternating poly(A) site usage. APA can thus result in changes in RNA processing, such as mRNA localization, stability, translation efficiency, and sometimes even in the localization of the encoded protein. It remains unclear, however, if and how APA is regulated by the circadian clock. To address this, we used an in silico approach and demonstrated in mouse liver that 57.4% of expressed genes undergo APA and each gene has 2.53 poly(A) sites on average. Among all expressed genes, 2.9% of genes alternate their poly(A) site usage with a circadian (i.e., approximately 24 hr) period. APA transcripts use distal sites with canonical poly(A) signals (PASs) more frequently; however, circadian APA transcripts exhibit less distinct usage preference between proximal and distal sites and use proximal sites more frequently. Circadian APA transcripts also harbor longer 3′UTRs, making them more susceptible to post-transcriptional regulation. Overall, our study serves as a platform to ultimately understand the mechanisms of circadian APA regulation.

Genome-wide CRISPR screen identifies HNRNPL as a prostate cancer dependency regulating RNA splicing

Alternative RNA splicing plays an important role in cancer. To determine which factors involved in RNA processing are essential in prostate cancer, we performed a genome-wide CRISPR/Cas9 knockout screen to identify the genes that are required for prostate cancer growth. Functional annotation defined a set of essential spliceosome and RNA binding protein (RBP) genes, including most notably heterogeneous nuclear ribonucleoprotein L (HNRNPL). We defined the HNRNPL-bound RNA landscape by RNA immunoprecipitation coupled with next-generation sequencing and linked these RBP-RNA interactions to changes in RNA processing. HNRNPL directly regulates the alternative splicing of a set of RNAs, including those encoding the androgen receptor, the key lineage-specific prostate cancer oncogene. HNRNPL also regulates circular RNA formation via back splicing. Importantly, both HNRNPL and its RNA targets are aberrantly expressed in human prostate tumors, supporting their clinical relevance. Collectively, our data reveal HNRNPL and its RNA clients as players in prostate cancer growth and potential therapeutic targets.

Widespread Shortening of 3' Untranslated Regions and Increased Exon Inclusion Are Evolutionarily Conserved Features of Innate Immune Responses to Infection.

The contribution of pre-mRNA processing mechanisms to the regulation of immune responses remains poorly studied despite emerging examples of their role as regulators of immune defenses. We sought to investigate the role of mRNA processing in the cellular responses of human macrophages to live bacterial infections. Here, we used mRNA sequencing to quantify gene expression and isoform abundances in primary macrophages from 60 individuals, before and after infection with Listeria monocytogenes and Salmonella typhimurium. In response to both bacteria we identified thousands of genes that significantly change isoform usage in response to infection, characterized by an overall increase in isoform diversity after infection. In response to both bacteria, we found global shifts towards (i) the inclusion of cassette exons and (ii) shorter 3’ UTRs, with near-universal shifts towards usage of more upstream polyadenylation sites. Using complementary data collected in non-human primates, we show that these features are evolutionarily conserved among primates. Following infection, we identify candidate RNA processing factors whose expression is associated with individual-specific variation in isoform abundance. Finally, by profiling microRNA levels, we show that 3’ UTRs with reduced abundance after infection are significantly enriched for target sites for particular miRNAs. These results suggest that the pervasive usage of shorter 3’ UTRs is a mechanism for particular genes to evade repression by immune-activated miRNAs. Collectively, our results suggest that dynamic changes in RNA processing may play key roles in the regulation of innate immune responses.

Cholinergic Surveillance over Hippocampal RNA Metabolism and Alzheimer's-Like Pathology.

The relationship between long-term cholinergic dysfunction and risk of developing dementia is poorly understood. Here we used mice with deletion of the vesicular acetylcholine transporter (VAChT) in the forebrain to model cholinergic abnormalities observed in dementia. Whole-genome RNA sequencing of hippocampal samples revealed that cholinergic failure causes changes in RNA metabolism. Remarkably, key transcripts related to Alzheimer's disease are affected. BACE1, for instance, shows abnormal splicing caused by decreased expression of the splicing regulator hnRNPA2/B1. Resulting BACE1 overexpression leads to increased APP processing and accumulation of soluble Aβ1-42. This is accompanied by age-related increases in GSK3 activation, tau hyperphosphorylation, caspase-3 activation, decreased synaptic markers, increased neuronal death, and deteriorating cognition. Pharmacological inhibition of GSK3 hyperactivation reversed deficits in synaptic markers and tau hyperphosphorylation induced by cholinergic dysfunction, indicating a key role for GSK3 in some of these pathological changes. Interestingly, in human brains there was a high correlation between decreased levels of VAChT and hnRNPA2/B1 levels with increased tau hyperphosphorylation. These results suggest that changes in RNA processing caused by cholinergic loss can facilitate Alzheimer's-like pathology in mice, providing a mechanism by which decreased cholinergic tone may increase risk of dementia.

MTHFSD and DDX58 are novel RNA-binding proteins abnormally regulated in amyotrophic lateral sclerosis.

Tar DNA-binding protein 43 (TDP-43) is an RNA-binding protein normally localized to the nucleus of cells, where it elicits functions related to RNA metabolism such as transcriptional regulation and alternative splicing. In amyotrophic lateral sclerosis, TDP-43 is mislocalized from the nucleus to the cytoplasm of diseased motor neurons, forming ubiquitinated inclusions. Although mutations in the gene encoding TDP-43, TARDBP, are found in amyotrophic lateral sclerosis, these are rare. However, TDP-43 pathology is common to over 95% of amyotrophic lateral sclerosis cases, suggesting that abnormalities of TDP-43 play an active role in disease pathogenesis. It is our hypothesis that a loss of TDP-43 from the nucleus of affected motor neurons in amyotrophic lateral sclerosis will lead to changes in RNA processing and expression. Identifying these changes could uncover molecular pathways that underpin motor neuron degeneration. Here we have used translating ribosome affinity purification coupled with microarray analysis to identify the mRNAs being actively translated in motor neurons of mutant TDP-43(A315T) mice compared to age-matched non-transgenic littermates. No significant changes were found at 5 months (presymptomatic) of age, but at 10 months (symptomatic) the translational profile revealed significant changes in genes involved in RNA metabolic process, immune response and cell cycle regulation. Of 28 differentially expressed genes, seven had a ≥ 2-fold change; four were validated by immunofluorescence labelling of motor neurons in TDP-43(A315T) mice, and two of these were confirmed by immunohistochemistry in amyotrophic lateral sclerosis cases. Both of these identified genes, DDX58 and MTHFSD, are RNA-binding proteins, and we show that TDP-43 binds to their respective mRNAs and we identify MTHFSD as a novel component of stress granules. This discovery-based approach has for the first time revealed translational changes in motor neurons of a TDP-43 mouse model, identifying DDX58 and MTHFSD as two TDP-43 targets that are misregulated in amyotrophic lateral sclerosis.

Rodent Models of Amyotrophic Lateral Sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a motor neuron disease affecting upper and lower motor neurons in the central nervous system. Patients with ALS develop extensive muscle wasting and atrophy leading to paralysis and death 3 to 5 years after disease onset. The condition may be familial (fALS 10%) or sporadic ALS (sALS, 90%). The large majority of fALS cases are due to genetic mutations in the Superoxide dismutase 1 gene (SOD1, 15% of fALS) and repeat nucleotide expansions in the gene encoding C9ORF72 (∼ 40% to 50% of fALS and ∼ 10% of sALS). Studies suggest that ALS is mediated through aberrant protein homeostasis (i.e., ER stress and autophagy) and/or changes in RNA processing (as in all non-SOD1-mediated ALS). In all of these cases, animal models suggest that the disorder is mediated non-cell autonomously, i.e., not only motor neurons are involved, but glial cells including microglia, astrocytes, and oligodendrocytes, and other neuronal subpopulations are also implicated in the pathogenesis. Provided in this unit is a review of ALS rodent models, including discussion of their relative advantages and disadvantages. Emphasis is placed on correlating the model phenotype with the human condition and the utility of the model for defining the disease process. Information is also presented on RNA processing studies in ALS research, with particular emphasis on the newest ALS rodent models.

Staphylococcus aureus Small Colony Variants (SCVs): a road map for the metabolic pathways involved in persistent infections.

Persistent and relapsing infections, despite apparently adequate antibiotic therapy, occur frequently with many pathogens, but it is an especially prominent problem with Staphylococcus aureus infections. For the purposes of this review, persistence will encompass both of the concepts of long term survival within the host, including colonization, and the concept of resisting antibiotic therapy even when susceptible in the clinical microbiology laboratory. Over the past two decades, the mechanisms whereby bacteria achieve persistence are slowly being unraveled. S. aureus small colony variants (SCVs) are linked to chronic, recurrent, and antibiotic-resistant infections, and the study of SCVs has contributed significantly to understanding of persistence. In our earlier work, defects in electron transport and thymidylate biosynthesis were linked to the development of the SCV phenotype (reviewed in 2006), thus this work will be discussed only briefly. Since 2006, it has been found that persistent organisms including SCVs are part of the normal life cycle of bacteria, and often they arise in response to harsh conditions, e.g., antibiotics, starvation, host cationic peptides. Many of the changes found in these early SCVs have provided a map for the discovery mechanisms (pathways) for the development of persistent organisms. For example, changes in RNA processing, stringent response, toxin-antitoxin, ribosome protein L6 (RplF), and cold shock protein B (CspB) found in SCVs are also found in other persisters. In addition, many classic persister organisms also show slow growth, hence SCVs. Recent work on S. aureus USA300 has elucidated the impact of aerobic expression of arginine deiminase genes on its ability to chronically colonize the skin and survive in abscesses. S. aureus SCVs also express arginine deiminase genes aerobically as well. Thus, many pathways found activated in electron transport type of SCVs are also increased in persisters that have intact electron transport. Many of these changes in metabolism result in slow growth; hence, small colonies are formed. Another common theme is that slow growth is also associated with reduced expression of virulence factors and enhanced uptake/survival within host cells. These adaptations to survive within the host are rooted in responses that were required for organisms to survive in a harsh environment long before they were mammals on the earth.

What, where, and when: the importance of post-transcriptional regulation in the brain

Until recently, RNA metabolism has been considered a purely academic topic that kept a small group of molecular biologists busy. Thanks to many emerging techniques—mainly, but not exclusively—systems approaches that employ high-throughput sequencing, such as UV cross-linking and immunoprecipitation (CLIP), ribosome profiling and RNAseq, we gained major new insights into the importance of RNA metabolism for brain function, as well as malfunction (Darnell, 2013). In that context, the brain presents a particularly fascinating diversity of post-transcriptional gene regulation through many new and recently discovered mechanisms. Neurons exhibit remarkably rich molecular repertoires that match their complex morphologies and functions. RNA is often localized to various subcompartments in order to exert specific local functions. Moreover, dynamic changes in RNA processing and turnover provide powerful mechanisms for neuronal plasticity. The central importance of these mechanisms is highlighted by the severe neurological disorders associated with defects in post-transcriptional processing functions in the brain.

The Eleven-Nineteen Lysine-rich Leukemia Gene (ELL2) Influences the Histone H3 Protein Modifications Accompanying the Shift to Secretory Immunoglobulin Heavy Chain mRNA Production

In plasma cells, immunoglobulin heavy chain (IgH) secretory-specific mRNA is made in high abundance as a result of both increased promoter proximal poly(A) site choice and weak splice-site skipping. Ell2, the eleven-nineteen lysine rich leukemia gene, is a transcription elongation factor that is induced ∼6-fold in plasma cells and has been shown to drive secretory-specific mRNA production. Reducing ELL2 by siRNA, which reduced processing to the secretion-specific poly(A) site, also influenced the methylations of histone H3K4 and H3K79 on the IgH gene and impacted positive transcription factor b (pTEFb), Ser-2 carboxyl-terminal phosphorylation, and polyadenylation factor additions to RNA polymerase II. The multiple lineage leukemia gene (MLL) and Dot1L associations with the IgH gene were also impaired in the absence of ELL2. To investigate the link between histone modifications, transcription elongation, and alternative RNA processing in IgH mRNA production, we performed chromatin immunoprecipitation on cultured mouse B and plasma cells bearing the identical IgH γ2a gene. In the plasma cells, as compared with the B cells, the H3K4 and H3K79 methylations extended farther downstream, past the IgH enhancer to the end of the transcribed region. Thus the downstream H3K4 and H3K79 methylation of the IgH associated chromatin in plasma cells is associated with increased polyadenylation and exon skipping, resulting from the actions of ELL2 transcription elongation factor.

SplicerAV: a tool for mining microarray expression data for changes in RNA processing

BackgroundOver the past two decades more than fifty thousand unique clinical and biological samples have been assayed using the Affymetrix HG-U133 and HG-U95 GeneChip microarray platforms. This substantial repository has been used extensively to characterize changes in gene expression between biological samples, but has not been previously mined en masse for changes in mRNA processing. We explored the possibility of using HG-U133 microarray data to identify changes in alternative mRNA processing in several available archival datasets.ResultsData from these and other gene expression microarrays can now be mined for changes in transcript isoform abundance using a program described here, SplicerAV. Using in vivo and in vitro breast cancer microarray datasets, SplicerAV was able to perform both gene and isoform specific expression profiling within the same microarray dataset. Our reanalysis of Affymetrix U133 plus 2.0 data generated by in vitro over-expression of HRAS, E2F3, beta-catenin (CTNNB1), SRC, and MYC identified several hundred oncogene-induced mRNA isoform changes, one of which recognized a previously unknown mechanism of EGFR family activation. Using clinical data, SplicerAV predicted 241 isoform changes between low and high grade breast tumors; with changes enriched among genes coding for guanyl-nucleotide exchange factors, metalloprotease inhibitors, and mRNA processing factors. Isoform changes in 15 genes were associated with aggressive cancer across the three breast cancer datasets.ConclusionsUsing SplicerAV, we identified several hundred previously uncharacterized isoform changes induced by in vitro oncogene over-expression and revealed a previously unknown mechanism of EGFR activation in human mammary epithelial cells. We analyzed Affymetrix GeneChip data from over 400 human breast tumors in three independent studies, making this the largest clinical dataset analyzed for en masse changes in alternative mRNA processing. The capacity to detect RNA isoform changes in archival microarray data using SplicerAV allowed us to carry out the first analysis of isoform specific mRNA changes directly associated with cancer survival.

SplicerAV: A tool for mining microarray expression data for changes in RNA processing

SplicerAV: A tool for mining microarray expression data for changes in RNA processing

Increased Phosphorylation of the Carboxyl-Terminal Domain of RNA Polymerase II and Loading of Polyadenylation and Cotranscriptional Factors Contribute to Regulation of the Ig Heavy Chain mRNA in Plasma Cells

B cells produce Ig H chain (IgH) mRNA and protein, primarily of the membrane-bound specific form. Plasma cells produce 20- to 50-fold higher amounts of IgH mRNA, most processed to the secretory specific form; this shift is mediated by substantial changes in RNA processing but only a small increase in IgH transcription rate. We investigated RNA polymerase II (RNAP-II) loading and phosphorylation of its C-terminal domain (CTD) on the IgG2a H chain gene, comparing two mouse cell lines representing B (A20) and plasma cells (AxJ) that express the identical H chain gene whose RNA is processed in different ways. Using chromatin immunoprecipitation and real-time PCR, we detected increased RNAP-II and Ser-2 and Ser-5 phosphorylation of RNAP-II CTD close to the IgH promoter in plasma cells. We detected increased association of several 3' end-processing factors, ELL2 and PC4, at the 5' end of the IgH gene in AxJ as compared with A20 cells. Polymerase progress and factor associations were inhibited by 5,6-dichlorobenzimidazole riboside, a drug that interferes with the addition of the Ser-2 to the CTD of RNAP-II. Taken together, these data indicate a role for CTD phosphorylation and polyadenylation/ELL2/PC4 factor loading on the polymerase in the choice of the secretory poly(A) site for the IgH gene.