Alternative Transcription Start Sites Research Articles

Functional Role of Linc-RNAs in Multiple Myeloma: Linc-MIR17HG Affects Fatty Acid Biosynthesis Via transcriptional Regulation of ACC1 with Potential Therapeutic Implications

Besides the well described function of RNA to produce proteins, a large volume of transcribed product has non-coding function. A recent analysis of RNA repertoire has identified a family of non-coding transcripts with sequence longer than 200 nucleotides, the long intergenic non-coding RNAs (lincRNAs). Although lincRNAs have been considered to provide regulatory functions, their precise role in cellular biology remains unclear. Using our RNA-seq data from 360 newly-diagnosed patients and 18 normal plasma cells, we have recently described the landscape of lincRNAs in multiple myeloma (MM) and reported their role as independent risk predictors for survival outcome.We have now studied the functional role of a lincRNA, the miR-17-92 primary precursor linc-MIR17HG, present in our lincRNA profile and highly correlated with overall survival in MM. We observe that inhibition of linc-MIR17HG by antisense LNA GapmeRs (n=2) leads to apoptosis in 12 genotypically distinct MM cell lines as well as in 13 primary patient MM cells. These effects are not fully rescued by expression of miR-17-92 microRNAs, suggesting a distinct biological function for linc-MIR17HG in MM. We therefore performed gene expression profile in 2 MM cell lines (AMO1 and NCI-H929) and in 2 primary patient MM cells after short-term suppression of linc-MIR17HG; and, at these early time points (18-36h), we found significant downregulation of a subset of genes (FC>2; p<0.05) including ACC1, EXT1, EPT1, ANO6, CCDC91 and KIA1109, but not miR-17-92 microRNAs. These transcriptional changes were validated by qRT-PCR in MM cell lines and primary MM cells exposed to different LNA GapmeRs targeting linc-MIR17HG with a non-overlapping spectrum of off-target effects. Importantly, our RNA-seq analysis of 360 newly-diagnosed MM patients from IFM/DFCI 2009 clinical study showed that expression of linc-MIR17HG strongly correlated with the expression of each of these genes (R2>0.4; p<0.01) in MM patients, further suggesting a regulatory function by linc-MIR17HG at transcriptional level. Using CRISPR interference (CRISPRi), we have also identified that the linc-MIR17HG with transcriptional regulatory functions is not produced from the canonical transcript isoforms MIR17HG-201/-203; these isoforms, rather, appear to be involved in production of microRNAs, leaving an alternative transcription start site usage as possible source for the transcriptional regulator isoform(s).We next investigated whether regulation of these early targets may contribute to the activity of linc-MIR17HG. We performed a RNAi-based screening in 2 MM cells lines (AMO1 and NCI-H929) by silencing each of the linc-MIR17HG downstream target genes with at least 2 different highly-specific siRNAs. This approach revealed that silencing of acetyl-CoA carboxylase 1 (ACC1, also known as ACACA), a gene encoding the limiting enzyme in the biosynthesis of fatty acids, significantly affects MM cell growth and viability. These results were validated using stable knock-down via shRNAs, confirming ACC1 as a novel vulnerability in MM. These results provide a molecular basis for reported role of fatty acid metabolism in MM cell growth and survival. We have now evaluated two orally available inhibitors of ACC1 activity, ND-630 and ND-646, in a panel of 10 MM cell lines, an report a potent time- and dose-dependent anti-proliferative effect. The activity of these inhibitors and linc-MIR17HG on fatty acid biosynthesis in MM cells is under investigation and will be presented.We have also begun to investigate molecular pathway used by linc-MIR17HG to modulate ACC1 function. Our preliminary data suggest that linc-MIR17HG may function as a scaffold between MYC and the E-box motifs present on ACC1 intronic sequences, facilitating MYC binding and its transcriptional activity.In conclusion, we highlight a transcriptional regulatory activity of a lincRNA in MM with significant functional impact that can be therapeutically translated. DisclosuresAnderson:Bristol Myers Squibb: Consultancy; Millennium Takeda: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; OncoPep: Equity Ownership, Other: Scientific founder; C4 Therapeutics: Equity Ownership, Other: Scientific founder; Celgene: Consultancy. Munshi:OncoPep: Other: Board of director.

WebHERV: A Web Server for the Computational Investigation of Gene Expression Associated With Endogenous Retrovirus-Like Sequences.

More than eight percent of the human genome consists of human endogenous retroviruses (HERVs). Typically, the expression of HERVs is repressed, but varying activities of HERVs have been observed in diseases ranging from cancer to neuro-degeneration. Such activities can include the transcription of HERV-derived open reading frames, which can be translated into proteins. However, as a consequence of mutations that disrupt open reading frames, most HERV-like sequences have lost their protein-coding capacity. Nevertheless, these loci can still influence the expression of adjacent genes and, hence, mediate biological effects. Here, we present WebHERV (http://calypso.informatik.uni-halle.de/WebHERV/), a web server that enables the computational prediction of active HERV-like sequences in the human genome based on a comparison of genome coordinates of expressed sequences uploaded by the user and genome coordinates of HERV-like sequences stored in the specialized key-value store DRUMS. Using WebHERV, we predicted putative candidates of active HERV-like sequences in Hodgkin lymphoma (HL) cell lines, validated one of them by a modified SMART (switching mechanism at 5′ end of RNA template) technique, and identified a new alternative transcription start site for cytochrome P450, family 4, subfamily Z, polypeptide 1 (CYP4Z1).

ProteomeGenerator: A Framework for Comprehensive Proteomics Based on de Novo Transcriptome Assembly and High-Accuracy Peptide Mass Spectral Matching.

Modern mass spectrometry now permits genome-scale and quantitative measurements of biological proteomes. However, analysis of specific specimens is currently hindered by the incomplete representation of biological variability of protein sequences in canonical reference proteomes and the technical demands for their construction. Here, we report ProteomeGenerator, a framework for de novo and reference-assisted proteogenomic database construction and analysis based on sample-specific transcriptome sequencing and high-accuracy mass spectrometry proteomics. This enables the assembly of proteomes encoded by actively transcribed genes, including sample-specific protein isoforms resulting from non-canonical mRNA transcription, splicing, or editing. To improve the accuracy of protein isoform identification in non-canonical proteomes, ProteomeGenerator relies on statistical target-decoy database matching calibrated using sample-specific controls. Its current implementation includes automatic integration with MaxQuant mass spectrometry proteomics algorithms. We applied this method for the proteogenomic analysis of splicing factor SRSF2 mutant leukemia cells, demonstrating high-confidence identification of non-canonical protein isoforms arising from alternative transcriptional start sites, intron retention, and cryptic exon splicing as well as improved accuracy of genome-scale proteome discovery. Additionally, we report proteogenomic performance metrics for current state-of-the-art implementations of SEQUEST HT, MaxQuant, Byonic, and PEAKS mass spectral analysis algorithms. Finally, ProteomeGenerator is implemented as a Snakemake workflow within a Singularity container for one-step installation in diverse computing environments, thereby enabling open, scalable, and facile discovery of sample-specific, non-canonical, and neomorphic biological proteomes.

Whole exome sequencing reveals novel NOV and DCAF13 variants in a Chinese pedigree with familial cortical myoclonic tremor with epilepsy

ObjectiveWe report a large new family of familial cortical myoclonic tremor with epilepsy(FCMTE) from China and identify the possible causative gene(s) for the family. MethodWhole exome sequencing of blood genomic DNA from 4 patients and 2 unaffected family members were performed. Detected variants and their cosegregation were confirmed by Sanger sequencing. ResultsWe identified c.20 G > C variant in the DCAF13 gene and c.983 T > C variant in the NOV gene cosegregating in the family. There was no additional cross-over in the family to narrow to one gene. The two DCAF13 and NOV gene variants are located on 8q23.3 and 8q24.12, which is consistent with the location 8q23.3–q24.13 reported previously for a group of Japanese families. The DCAF13 variant is located in alternative transcription start site(TSS) and the function of alternative TSS is unknown. The missense NOV variant is near the C terminus in a site that is highly conserved across species. It was predicted to be deleterious on protein function. ConclusionsIn this study, we identify two novel variants in the DCAF13 and NOV genes associated with FCMTE in Asian populations. The interval between two variants is 15.6Mb, which is very close to each other. Future studies of additional families with this phenotype are warranted to confirm whether it is rare bigenic or monogenic inheritance.

Abstract SY31-03: Reuse of genomics data and lessons for cancer research

Abstract Making data and materials used in scientific research available to others is not only a prerequisite for enabling reproducible science and maximizing the return on funding, it is also the very basis of scientific progress, allowing other scientists to build on the previous work of their colleagues (1). In life sciences this principle has been well established since the 1980s, when scientific journals started making it a requirement, that X-ray crystallography and DNA sequence data supporting publications must be deposited in appropriate databases. In 1996, as a part of the Human Genome Project, it was agreed that all the sequence data would be released in publicly accessible databases within twenty-four hours after generation, known as Bermuda principles (2). With advent of new types of data, such as microarray data, it was soon recognized that not only the sequence or microarray data are important, but also the standards how these data are represented and the information about the samples and experiments (3). However, to enable data sharing, a properly built and funded infrastructure is needed (4). Without public data resources, such as Ensembl, UniProt, and Expression Atlas, that add value to molecular data archives, modern life sciences research would be hard to imagine (5-7). The data-sharing mentality is now so firmly embedded in the ethos of life sciences that scientists working in the field struggle to imagine that in other science disciplines the mentality may be different. Data sharing in medical research is a more complex and difficult problem due to multiple reasons, including the need for data security and the patient's confidentiality, complexity of representing health records, and diversity of national legislations. However, there is also an increasing realization that sharing biomedical research data, which is now facilitated by the use of electronic health records, can be an important accelerator of biomedical research (7). Cancer researchers are at the forefront of the data-sharing approach in biomedical research. The International Cancer Genome Consortium is completing the sequencing of genomes, transcriptomes, and epigenomes of over 20,000 patients, making all the data alongside the essential clinical information available to researchers (8). About 10% of these genomes and transcriptomes have been reanalyzed in a standardized way by the Pan-cancer Analysis of the Whole Genomes (PCAWG) group. The PCAWG project provides an important demonstration how data integration can accelerate biomedical research. In this talk I will particularly concentrate on lessons learned from integration of genome, transcriptome, and clinical data of this project. Although at the time of writing this abstract, some of the PCAWG transcriptome analysis is still being finalized, it is already clear that integrating heterogeneous types of data from heterogeneous cancer types provides new insights about this disease, and also that such integrative analysis is challenging (9-13). I will also describe some of the experience in building infrastructure for data integration and sharing at the European Bioinformatics Institute (EMBL-EBI) and some of the data resources provided by EMBL-EBI (14,15), particularly concentrating on cancer genomics data and the benefits that data sharing brings to cancer research.

Abstract 3494: Downregulation of SREBPs inhibited colon cancer cell growth and tumor initiation by alteration of metabolism

Abstract Sterol regulatory element binding proteins (SREBPs) belong to a family of transcription factors that regulate the expression of genes required for the synthesis of fatty acids and cholesterol. There are three SREBP isoforms, SREBP1a, SREBP1c and SREBP2, have been identified in mammalian cells. SREBP1a and SREBP1c are derived from a single gene through the use of alternative transcription start sites. Increasing evidence suggests that increased lipogenesis is needed for cancer cells to accommodate high rates of proliferation. Activation of SREBP1 and enhanced expression of its target genes have been observed in different types of human cancers. However, the functional importance of SREBPs in colon cancer remains to be fully elucidated. In this study, we investigated the role of SREBP-mediated lipid biosynthesis in promoting tumor growth and initiation by regulating cellular metabolism in colon cancer. Knockdown of SREBP-1 or SREBP-2 decreased the expression of target genes required for lipid biosynthesis and levels of fatty acids and triacylglycerides in colon cancer cells. Bioenergetic analysis using Seahorse XF96 Extracelluar Flux Analyzer revealed that silencing SREBP-1 or SREBP-2 expression reduced the mitochondrial respiration, glycolysis, as well as fatty acid oxidation suggesting an inhibition of cellular metabolism. As a result, the rate of cell proliferation and the ability of cancer cells to form tumor spheroids in suspension were decreased. Similar results were obtained in colon cancer cells in which SCAP, an upstream protease responsible for activating SREBP-1 and SREBP-2, was silenced. Mechanistically, downregulation of SREBPs inhibited the activation of AMPK and Akt signaling. Furthermore, knockdown of SREBP-1 or SREBP-2 inhibited xenograft tumor growth in vivo and significantly decreased the expression of genes associated cancer stem cells. Taken together, our findings establish the molecular basis of SREBPs-dependent metabolic regulation and provide a rationale for targeting fatty acid synthesis as a promising approach in colon cancer treatment. Citation Format: Yang-an Wen, Xiaopeng Xiong, Yekaterina Zaytseva1, Tianyan Gao. Downregulation of SREBPs inhibited colon cancer cell growth and tumor initiation by alteration of metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3494.

Transcripts from downstream alternative transcription start sites evade uORF-mediated inhibition of gene expression in Arabidopsis

Plants adapt to alterations in light conditions by controlling their gene expression profiles. Expression of light-inducible genes is transcriptionally induced by transcription factors such as HY5. However, few detailed analyses have been carried out on the control of transcription start sites (TSSs). Of the various wavelengths of light, it is blue light (BL) that regulates physiological responses such as hypocotyl elongation and flowering time. To understand how gene expression is controlled not only by transcript abundance but also by TSS selection, we examined genome-wide TSS profiles in Arabidopsis seedlings after exposure to BL irradiation following initial growth in the dark. Thousands of genes use multiple TSSs, and some transcripts have upstream ORFs (uORFs) that take precedence over the main ORF (mORF) encoding proteins. The uORFs often function as translation inhibitors of the mORF or as triggers of nonsense-mediated mRNA decay (NMD). Transcription from TSSs located downstream of the uORFs in 220 genes is enhanced by BL exposure. This type of regulation is found in HY5 and HYH, major regulators of light-dependent gene expression. Translation efficiencies of the genes showing enhanced usage of these TSSs increased upon BL exposure. We also show that transcripts from TSSs upstream of uORFs in 45 of the 220 genes, including HY5, accumulated in a mutant of NMD. These results suggest that BL controls gene expression not only by enhancing transcriptions but also by choosing the TSS, and transcripts from downstream TSSs evade uORF-mediated inhibition to ensure high expression of light-regulated genes.

Characterization of the Regulation and Function of Tetratricopeptide 39c (Ttc39c) in Skeletal Muscle

Ttc39c has been identified as a novel gene in skeletal muscle that is differentially regulated in response to neurogenic atrophy in wild‐type (WT) mice compared to Muscle RING Finger 1 (MuRF1) knockout (KO) mice. MuRF1 is a ubiquitin E3 ligase that is known to be an important mediator of muscle wasting; however, more recent research suggests that MuRF1 may also function as a regulator of atrophy‐induced gene expression. Quantitative PCR and Western blot analysis determined that Ttc39c is expressed in both proliferating and differentiated muscle cells. Furthermore, comparison of Ttc39c expression in undifferentiated and differentiated C2C12 cells demonstrated that Ttc39c levels are elevated in proliferating myoblasts, but decrease in differentiated myotubes. The transcriptional regulation of two Ttc39c isoforms with alternative transcriptional start sites were examined by cloning the unique promoter regions of each transcript. The Ttc39c reporter gene constructs were transfected into muscle cells and confirmed to have significant transcriptional activity in cultured muscle cells and are transcriptionally responsive to ectopic expression of myogenic regulatory factors (MRF). Furthermore, conserved E‐box elements in the proximal promoter regions of both Ttc39c transcripts were identified, mutated, and analyzed for their role in the transcriptional regulation of Ttc39c expression. The mutation of the conserved E‐box sequences rendered the Ttc39c reporter genes inactive, suggesting that these elements are potentially necessary for Ttc39c expression. Finally, immunoprecipitation (IP) of Ttc39c from C2C12 cells resulted in the co‐IP of numerous binding partners, which are currently being identified by proteomic analysis in the hope of elucidating a potential function for Ttc39c in skeletal muscle based on identified binding partners. Interestingly, ectopic expression of Ttc39c in C2C12 mouse myoblasts resulted in delayed cell fusion and reduction in myotube formation. The identification and characterization of novel genes that are activated during neurogenic atrophy helps improve our understanding of the molecular genetic events that result in muscle wasting and could eventually lead to new therapeutic targets in the future.Support or Funding InformationThe work was support by University of North Florida Transformational Learning Opportunity grants to D.W.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Expression of novel "LOCGEF" isoforms of ARHGEF18 in eosinophils.

Genomic, transcriptomic and proteomic databases indicate that the N-terminal 322 residues encoded by the presumptive LOC100996504 gene, which is adjacent to the ARHGEF18 guanine nucleotide exchange factor gene on chromosome 19, constitute the N-terminal portion of a 1361-residue isoform of ARHGEF18, dubbed LOCGEF-X3. LOCGEF-X3 arises from the use of a leukocyte-specific alternative transcriptional start site and splicing that bypasses the initial noncoding exon of the canonical 1015-residue ARHGEF18 isoform, p114. Eosinophil LOCGEF-X3 was amplified and cloned, recombinant LOCGEF-X3 was expressed, and anti-ARHGEF18 antibody was found to recognize a band in immunoblots of eosinophil lysates that co-migrates with recombinant LOCGEF-X3. PCR of eosinophils revealed minor amounts of transcripts for X4 and X5 isoforms of LOCGEF that arise from differential splicing and differ from the X3 isoform at their extreme N-termini. No p114 transcript or protein band was detected in eosinophils. Immunostaining with anti-ARHGEF18 antibody revealed relocalization of LOCGEF and RHOA from the periphery of round unstimulated eosinophils to the 2 poles of eosinophils polarized by treatment with IL5, CCL11, or IL33 in suspension. Canonical p114 ARHGEF18 has been implicated in maintenance of epithelial cell polarity. We suggest that the "LOC" portion of LOCGEF, which is unlike any other protein domain, has unique functions in control of polarity in activated eosinophils and other leukocytes.

SEASTAR: systematic evaluation of alternative transcription start sites in RNA.

Alternative first exons diversify the transcriptomes of eukaryotes by producing variants of the 5′ Untranslated Regions (5′UTRs) and N-terminal coding sequences. Accurate transcriptome-wide detection of alternative first exons typically requires specialized experimental approaches that are designed to identify the 5′ ends of transcripts. We developed a computational pipeline SEASTAR that identifies first exons from RNA-seq data alone then quantifies and compares alternative first exon usage across multiple biological conditions. The exons inferred by SEASTAR coincide with transcription start sites identified directly by CAGE experiments and bear epigenetic hallmarks of active promoters. To determine if differential usage of alternative first exons can yield insights into the mechanism controlling gene expression, we applied SEASTAR to an RNA-seq dataset that tracked the reprogramming of mouse fibroblasts into induced pluripotent stem cells. We observed dynamic temporal changes in the usage of alternative first exons, along with correlated changes in transcription factor expression. Using a combined sequence motif and gene set enrichment analysis we identified N-Myc as a regulator of alternative first exon usage in the pluripotent state. Our results demonstrate that SEASTAR can leverage the available RNA-seq data to gain insights into the control of gene expression and alternative transcript variation in eukaryotic transcriptomes.

Downregulation of SREBP inhibits tumor growth and initiation by altering cellular metabolism in colon cancer

Sterol regulatory element-binding proteins (SREBPs) belong to a family of transcription factors that regulate the expression of genes required for the synthesis of fatty acids and cholesterol. Three SREBP isoforms, SREBP1a, SREBP1c, and SREBP2, have been identified in mammalian cells. SREBP1a and SREBP1c are derived from a single gene through the use of alternative transcription start sites. Here we investigated the role of SREBP-mediated lipogenesis in regulating tumor growth and initiation in colon cancer. Knockdown of either SREBP1 or SREBP2 decreased levels of fatty acids as a result of decreased expression of SREBP target genes required for lipid biosynthesis in colon cancer cells. Bioenergetic analysis revealed that silencing SREBP1 or SREBP2 expression reduced the mitochondrial respiration, glycolysis, as well as fatty acid oxidation indicating an alteration in cellular metabolism. Consequently, the rate of cell proliferation and the ability of cancer cells to form tumor spheroids in suspension culture were significantly decreased. Similar results were obtained in colon cancer cells in which the proteolytic activation of SREBP was blocked. Importantly, knockdown of either SREBP1 or SREBP2 inhibited xenograft tumor growth in vivo and decreased the expression of genes associated with cancer stem cells. Taken together, our findings establish the molecular basis of SREBP-dependent metabolic regulation and provide a rationale for targeting lipid biosynthesis as a promising approach in colon cancer treatment.

Mitochondrial tyrosyl-DNA phosphodiesterase 2 and its TDP2S short isoform.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs abortive topoisomerase II cleavage complexes. Here, we identify a novel short isoform of TDP2 (TDP2S) expressed from an alternative transcription start site. TDP2S contains a mitochondrial targeting sequence, contributing to its enrichment in the mitochondria and cytosol, while full-length TDP2 contains a nuclear localization signal and the ubiquitin-associated domain in the N-terminus. Our study reveals that both TDP2 isoforms are present and active in the mitochondria. Comparison of isogenic wild-type (WT) and TDP2 knockout (TDP2-/-/-) DT40 cells shows that TDP2-/-/- cells are hypersensitive to mitochondrial-targeted doxorubicin (mtDox), and that complementing TDP2-/-/- cells with human TDP2 restores resistance to mtDox. Furthermore, mtDox selectively depletes mitochondrial DNA in TDP2-/-/- cells. Using CRISPR-engineered human cells expressing only the TDP2S isoform, we show that TDP2S also protects human cells against mtDox. Finally, lack of TDP2 in the mitochondria reduces the mitochondria transcription levels in two different human cell lines. In addition to identifying a novel TDP2S isoform, our report demonstrates the presence and importance of both TDP2 isoforms in the mitochondria.

Alternative start and termination sites of transcription drive most transcript isoform differences across human tissues.

Most human genes generate multiple transcript isoforms. The differential expression of these isoforms can help specify cell types. Diverse transcript isoforms arise from the use of alternative transcription start sites, polyadenylation sites and splice sites; however, the relative contribution of these processes to isoform diversity in normal human physiology is unclear. To address this question, we investigated cell type-dependent differences in exon usage of over 18 000 protein-coding genes in 23 cell types from 798 samples of the Genotype-Tissue Expression Project. We found that about half of the expressed genes displayed tissue-dependent transcript isoforms. Alternative transcription start and termination sites, rather than alternative splicing, accounted for the majority of tissue-dependent exon usage. We confirmed the widespread tissue-dependent use of alternative transcription start sites in a second, independent dataset, Cap Analysis of Gene Expression data from the FANTOM consortium. Moreover, our results indicate that most tissue-dependent splicing involves untranslated exons and therefore may not increase proteome complexity. Thus, alternative transcription start and termination sites are the principal drivers of transcript isoform diversity across tissues, and may underlie the majority of cell type specific proteomes and functions.

Alternative transcription start site selection in Mr-OPY2 controls lifestyle transitions in the fungus Metarhizium robertsii

Metarhizium robertsii is a versatile fungus with saprophytic, plant symbiotic and insect pathogenic lifestyle options. Here we show that M. robertsii mediates the saprophyte-to-insect pathogen transition through modulation of the expression of a membrane protein, Mr-OPY2. Abundant Mr-OPY2 protein initiates appressorium formation, a prerequisite for infection, whereas reduced production of Mr-OPY2 elicits saprophytic growth and conidiation. The precise regulation of Mr-OPY2 protein production is achieved via alternative transcription start sites. During saprophytic growth, a single long transcript is produced with small upstream open reading frames in its 5′ untranslated region. Increased production of Mr-OPY2 protein on host cuticle is achieved by expression of a transcript variant lacking a small upstream open reading frame that would otherwise inhibit translation of Mr-OPY2. RNA-seq and qRT-PCR analyses show that Mr-OPY2 is a negative regulator of a transcription factor that we demonstrate is necessary for appressorial formation. These findings provide insights into the mechanisms regulating fungal lifestyle transitions.

Global profiling of Rbm24 bound RNAs uncovers a multi-tasking RNA binding protein.

RNA binding proteins serve as critical molecular switches in a multitude of post-transcriptional regulatory processes. In the heart and muscles, the tissue specific RNA binding protein, Rbm24, is known to play important developmental roles via driving different post-transcriptional processes. Nonetheless, the currently identified molecular targets and regulatory pathways seem inadequate to completely explain the observed developmental effects upon Rbm24 knockdown/knockout. Here, by performing RNA Immunoprecipitation and coupling it to microarrays (RIP-Chip), we have generated an atlas of the mRNA binding repertoire of Rbm24. Further functional evaluation of its targets led to the elucidation of novel roles for Rbm24 in post-transcriptional processing, besides its already known roles in regulation of mRNA stability and alternative splicing. Interestingly, Rbm24 is found to cause the destabilization of Chrm2 via binding to an element in the coding region. In addition, Rbm24 is also found to have an uncharacterized role in driving the generation of isoforms with alternative transcriptional start sites. We have, for the first time, demonstrated that Rbm24 is a multi-tasking RNA binding protein capable of regulating its bound targets via a range of mechanisms.

Continuous Elution Proteoform Identification of Myelin Basic Protein by Superficially Porous Reversed-Phase Liquid Chromatography and Fourier Transform Mass Spectrometry.

Myelin basic protein (MBP) plays an important structural and functional role in the neuronal myelin sheath. Translated MBP exhibits extreme microheterogeneity with numerous alternative splice variants (ASVs) and post-translational modifications (PTMs) reportedly tied to central nervous system maturation, myelin stability, and the pathobiology of various de- and dys-myelinating disorders. Conventional bioanalytical tools cannot efficiently examine ASV and PTM events simultaneously, which limits understanding of the role of MBP microheterogeneity in human physiology and disease. To address this need, we report on a top-down proteomics pipeline that combines superficially porous reversed-phase liquid chromatography (SPLC), Fourier transform mass spectrometry (FTMS), data-independent acquisition (DIA) with nozzle-skimmer dissociation (NSD), and aligned data processing resources to rapidly characterize abundant MBP proteoforms within murine tissue. The three-tier proteoform identification and characterization workflow resolved four known MBP ASVs and hundreds of differentially modified states from a single 90 min SPLC-FTMS run on ∼0.5 μg of material. This included 323 proteoforms for the 14.1 kDa ASV alone. We also identified two novel ASVs from an alternative transcriptional start site (ATSS) of the MBP gene as well as a never before characterized S-acylation event linking palmitic acid, oleic acid, and stearic acid at C78 of the 17.125 kDa ASV.

Novel calpain families and novel mechanisms for calpain regulation in Aplysia.

Calpains are a family of intracellular proteases defined by a conserved protease domain. In the marine mollusk Aplysia californica, calpains are important for the induction of long-term synaptic plasticity and memory, at least in part by cleaving protein kinase Cs (PKCs) into constitutively active kinases, termed protein kinase Ms (PKMs). We identify 14 genes encoding calpains in Aplysia using bioinformatics, including at least one member of each of the four major calpain families into which metazoan calpains are generally classified, as well as additional truncated and atypical calpains. Six classical calpains containing a penta-EF-hand (PEF) domain are present in Aplysia. Phylogenetic analysis determined that these six calpains come from three separate classical calpain families. One of the classical calpains in Aplysia, AplCCal1, has been implicated in plasticity. We identify three splice cassettes and an alternative transcriptional start site in AplCCal1. We characterize several of the possible isoforms of AplCCal1 in vitro, and demonstrate that AplCCal1 can cleave PKCs into PKMs in a calcium-dependent manner in vitro. We also find that AplCCal1 has a novel mechanism of auto-inactivation through N-terminal cleavage that is modulated through its alternative transcriptional start site.

A Novel Functional Splice Variant of AKT3 Defined by Analysis of Alternative Splice Expression in HPV-Positive Oropharyngeal Cancers.

The incidence of HPV-related oropharyngeal squamous cell carcinoma (OPSCC) has increased more than 200% in the past 20 years. Recent genetic sequencing efforts have elucidated relevant genes in head and neck cancer, but HPV-related tumors have consistently shown few DNA mutations. In this study, we sought to analyze alternative splicing events (ASE) that could alter gene function independent of mutations. To identify ASE unique to HPV-related tumors, RNA sequencing was performed on 46 HPV-positive OPSCC and 25 normal tissue samples. A novel algorithm using outlier statistics on RNA-sequencing junction expression identified 109 splicing events, which were confirmed in a validation set from The Cancer Genome Atlas. Because the most common type of splicing event identified was an alternative start site (39%), MBD-seq genome-wide CpG methylation data were analyzed for methylation alterations at promoter regions. ASE in six genes showed significant negative correlation between promoter methylation and expression of an alternative transcriptional start site, including AKT3 The novel AKT3 transcriptional variant and methylation changes were confirmed using qRT-PCR and qMSP methods. In vitro silencing of the novel AKT3 variant resulted in significant growth inhibition of multiple head and neck cell lines, an effect not observed with wild-type AKT3 knockdown. Analysis of ASE in HPV-related OPSCC identified multiple alterations likely involved in carcinogenesis, including a novel, functionally active transcriptional variant of AKT3 Our data indicate that ASEs represent a significant mechanism of oncogenesis with untapped potential for understanding complex genetic changes that result in the development of cancer. Cancer Res; 77(19); 5248-58. ©2017 AACR.

Transcriptional profiling of Rickettsia prowazekii coding and non-coding transcripts during in vitro host-pathogen and vector-pathogen interactions

Natural pathogen transmission of Rickettsia prowazekii, the etiologic agent of epidemic typhus, to humans is associated with arthropods, including human body lice, ticks, and ectoparasites of eastern flying squirrel. Recently, we have documented the presence of small RNAs in Rickettsia species and expression of R. prowazekii sRNAs during infection of cultured human microvascular endothelial cells (HMECs), which represent the primary target cells during human infections. Bacterial noncoding transcripts are now well established as critical post-transcriptional regulators of virulence and adaptation mechanisms in varying host environments. Despite their importance, little is known about the expression profile and regulatory activities of R. prowazekii sRNAs (Rp_sRs) in different host cells encountered as part of the natural life-cycle. To investigate the sRNA expression profile of R. prowazekii during infection of arthropod host cells, we employed an approach combining in vitro infection, bioinformatics, RNA sequencing, and PCR-based quantitation. Global analysis of R. prowazekii transcriptome by strand-specific RNA sequencing enabled us to identify 67 cis-acting (antisense) and 26 trans-acting (intergenic) Rp_sRs expressed during the infection of Amblyomma americanum (AAE2) cells. Comparative evaluation of expression during R. prowazekii infection of HMECs and AAE2 cells by quantitative RT-PCR demonstrated significantly higher expression of four selected Rp_sRs in tick AAE2 cells. Examination of the coding transcriptome revealed differential up-regulation of >150 rickettsial genes in either HMECs or AAE2 cells and yielded evidence for host cell-dependent utilization of alternative transcription start sites by 18 rickettsial genes. Our results thus suggest noticeable differences in the expression of both Rp_sRs as well as the coding transcriptome and the exploitation of multiple transcription initiation sites for select genes during the infection of human endothelium and tick vector cells as the host and yield new insights into rickettsial virulence and transmission mechanisms.

Identification and prediction of alternative transcription start sites that generate rod photoreceptor-specific transcripts from ubiquitously expressed genes.

Transcriptome complexity is substantially increased by the use of multiple transcription start sites for a given gene. By utilizing a rod photoreceptor-specific chromatin signature, and the RefSeq database of established transcription start sites, we have identified essentially all known rod photoreceptor genes as well as a group of novel genes that have a high probability of being expressed in rod photoreceptors. Approximately half of these novel rod genes are transcribed into multiple mRNA and/or protein isoforms through alternative transcriptional start sites (ATSS), only one of which has a rod-specific epigenetic signature and gives rise to a rod transcript. This suggests that, during retina development, some genes use ATSS to regulate cell type and temporal specificity, effectively generating a rod transcript from otherwise ubiquitously expressed genes. Biological confirmation of the relationship between epigenetic signatures and gene expression, as well as comparison of our genome-wide chromatin signature maps with available data sets for retina, namely a ChIP-on-Chip study of Polymerase-II (Pol-II) binding sites, ChIP-Seq studies for NRL- and CRX- binding sites and DHS (University of Washington data, available on UCSC mouse Genome Browser as a part of ENCODE project) fully support our hypothesis and together accurately identify and predict an array of new rod transcripts. The same approach was used to identify a number of TSS that are not currently in RefSeq. Biological conformation of the use of some of these TSS suggests that this method will be valuable for exploring the range of transcriptional complexity in many tissues. Comparison of mouse and human genome-wide data indicates that most of these alternate TSS appear to be present in both species, indicating that our approach can be useful for identification of regulatory regions that might play a role in human retinal disease.