Trans-acting Factors Research Articles

HnRNP-A1 binds to the IRES of MELOE-1 antigen to promote MELOE-1 translation in stressed melanoma cells.

The major challenge in antigen‐specific immunotherapy of cancer is to select the most relevant tumor antigens to target. To this aim, understanding their mode of expression by tumor cells is critical. We previously identified a melanoma‐specific antigen, melanoma‐overexpressed antigen 1 (MELOE‐1)—coded for by a long noncoding RNA—whose internal ribosomal entry sequence (IRES)‐dependent translation is restricted to tumor cells. This restricted expression is associated with the presence of a broad‐specific T‐cell repertoire that is involved in tumor immunosurveillance in melanoma patients. In the present work, we explored the translation control of MELOE‐1 and provide evidence that heterogeneous nuclear ribonucleoprotein A1 (hnRNP‐A1) binds to the MELOE‐1 IRES and acts as an IRES trans‐activating factor (ITAF) to promote the translation of MELOE‐1 in melanoma cells. In addition, we showed that endoplasmic reticulum (ER) stress induced by thapsigargin, which promotes hnRNP‐A1 cytoplasmic translocation, enhances MELOE‐1 translation and recognition of melanoma cells by a MELOE‐1‐specific T‐cell clone. These findings suggest that pharmacological stimulation of stress pathways may enhance the efficacy of immunotherapies targeting stress‐induced tumor antigens such as MELOE‐1.

Analysis of the proximal promoter of the human colon-specific B4GALNT2 (Sda synthase) gene: B4GALNT2 is transcriptionally regulated by ETS1

Background: The Sda antigen and corresponding biosynthetic enzyme B4GALNT2 are primarily expressed in normal colonic mucosa and are down-regulated to a variable degree in colon cancer tissues. Although their expression profile is well studied, little is known about the underlying regulatory mechanisms. Methods: To clarify the molecular basis of Sda expression in the human gastrointestinal tract, we investigated the transcriptional regulation of the human B4GALNT2 gene. The proximal promoter region was delineated using luciferase assays and essential trans-acting factors were identified through transient overexpression and silencing of several transcription factors. Results: A short cis-regulatory region restricted to the −72 to +12 area upstream of the B4GALNT2 short-type transcript variant contained the essential promoter activity that drives the expression of the human B4GALNT2 regardless of the cell type. We further showed that B4GALNT2 transcriptional activation mostly requires ETS1 and to a lesser extent SP1. Conclusions: Results presented herein are expected to provide clues to better understand B4GALNT2 regulatory mechanisms.

Neoxanthin prevents H2O2-induced cytotoxicity in HepG2 cells by activating endogenous antioxidant signals and suppressing apoptosis signals.

The liver has a solid inbuilt antioxidant defense system to regulate oxidative stress. However, exposure to an excessive level of ROS causes liver injury. This study examined the cytoprotective effect of neoxanthin, a xanthophyll antioxidant molecule isolated from Solanum trilobatum in stress-induced HepG2 cells. The cytotoxic effect of H2O2 and cytoprotective potential of β-carotene, lutein, and neoxanthin was analyzed by WST-1 assay. The intracellular ROS level and mitochondrial membrane potential (MMP) were measured using DCFH-DA (2', 7'-dichlorofluorescin diacetate) and JC-10 MMP assay. The expression of anti-oxidant and apoptotic markers was measured by western blot analysis. Neoxanthin pretreatment exhibited better protection than β-carotene and lutein against cell death caused by H2O2. It significantly arrested H2O2-mediated elevation of intracellular ROS levels and protected MMP. The intracellular antioxidant enzymes HO-1 and SOD-2 were upregulated by neoxanthin pretreatment. Neoxanthin also activated the protein expression of redox-sensitive transactivation factors, Nrf2 and NF-kB. The cytoprotective effect of neoxanthin was associated with increased expression of the anti-apoptotic protein, Bcl-2 and decreased pro-apoptotic protein Bax. For the first time, our results demonstrate that neoxanthin offers adequate protection against stress-mediated cytotoxicity in hepatocytes by activating the intracellular antioxidant defense system and blocking apoptosis.

DDX21, a Host Restriction Factor of FMDV IRES-Dependent Translation and Replication.

In cells, the contributions of DEAD-box helicases (DDXs), without which cellular life is impossible, are of utmost importance. The extremely diverse roles of the nucleolar helicase DDX21, ranging from fundamental cellular processes such as cell growth, ribosome biogenesis, protein translation, protein–protein interaction, mediating and sensing transcription, and gene regulation to viral manipulation, drew our attention. We designed this project to study virus–host interactions and viral pathogenesis. A pulldown assay was used to investigate the association between foot-and-mouth disease virus (FMDV) and DDX21. Further insight into the DDX21–FMDV interaction was obtained through dual-luciferase, knockdown, overexpression, qPCR, and confocal microscopy assays. Our results highlight the antagonistic feature of DDX21 against FMDV, as it progressively inhibited FMDV internal ribosome entry site (IRES) -dependent translation through association with FMDV IRES domains 2, 3, and 4. To subvert this host helicase antagonism, FMDV degraded DDX21 through its non-structural proteins 2B, 2C, and 3C protease (3Cpro). Our results suggest that DDX21 is degraded during 2B and 2C overexpression and FMDV infection through the caspase pathway; however, DDX21 is degraded through the lysosomal pathway during 3Cpro overexpression. Further investigation showed that DDX21 enhanced interferon-beta and interleukin-8 production to restrict viral replication. Together, our results demonstrate that DDX21 is a novel FMDV IRES trans-acting factor, which negatively regulates FMDV IRES-dependent translation and replication.

Identification and Functional Characterization of Two Noncoding RNAs Transcribed from Putative Active Enhancers in Hepatocellular Carcinoma.

Enhancers have been conventionally perceived as cis-acting elements that provide binding sites for trans-acting factors. However, recent studies have shown that enhancers are transcribed and that these transcripts, called enhancer RNAs (eRNAs), have a regulatory function. Here, we identified putative eRNAs by profiling and determining the overlap between noncoding RNA expression loci and eRNA-associated histone marks such as H3K27ac and H3K4me1 in hepatocellular carcinoma (HCC) cell lines. Of the 132 HCC-derived noncoding RNAs, 74 overlapped with the eRNA loci defined by the FANTOM consortium, and 65 were located in the proximal regions of genes differentially expressed between normal and tumor tissues in TCGA dataset. Interestingly, knockdown of two selected putative eRNAs, THUMPD3-AS1 and LINC01572, led to downregulation of their target mRNAs and to a reduction in the proliferation and migration of HCC cells. Additionally, the expression of these two noncoding RNAs and target mRNAs was elevated in tumor samples in the TCGA dataset, and high expression was associated with poor survival of patients. Collectively, our study suggests that noncoding RNAs such as THUMPD3-AS1 and LINC01572 (i.e., putative eRNAs) can promote the transcription of genes involved in cell proliferation and differentiation and that the dysregulation of these noncoding RNAs can cause cancers such as HCC.

Defining the RBPome of primary T helper cells to elucidate higher-order Roquin-mediated mRNA regulation

Post-transcriptional gene regulation in T cells is dynamic and complex as targeted transcripts respond to various factors. This is evident for the Icos mRNA encoding an essential costimulatory receptor that is regulated by several RNA-binding proteins (RBP), including Roquin-1 and Roquin-2. Here, we identify a core RBPome of 798 mouse and 801 human T cell proteins by utilizing global RNA interactome capture (RNA-IC) and orthogonal organic phase separation (OOPS). The RBPome includes Stat1, Stat4 and Vav1 proteins suggesting unexpected functions for these transcription factors and signal transducers. Based on proximity to Roquin-1, we select ~50 RBPs for testing coregulation of Roquin-1/2 targets by induced expression in wild-type or Roquin-1/2-deficient T cells. Besides Roquin-independent contributions from Rbms1 and Cpeb4 we also show Roquin-1/2-dependent and target-specific coregulation of Icos by Celf1 and Igf2bp3. Connecting the cellular RBPome in a post-transcriptional context, we find contributions from multiple RBPs to the prototypic regulation of mRNA targets by individual trans-acting factors.

Zona Pellucida Genes and Proteins: Essential Players in Mammalian Oogenesis and Fertility.

All mammalian oocytes and eggs are surrounded by a relatively thick extracellular matrix (ECM), the zona pellucida (ZP), that plays vital roles during oogenesis, fertilization, and preimplantation development. Unlike ECM surrounding somatic cells, the ZP is composed of only a few glycosylated proteins, ZP1–4, that are unique to oocytes and eggs. ZP1–4 have a large region of polypeptide, the ZP domain (ZPD), consisting of two subdomains, ZP-N and ZP-C, separated by a short linker region, that plays an essential role in polymerization of nascent ZP proteins into crosslinked fibrils. Both subdomains adopt immunoglobulin (Ig)-like folds for their 3-dimensional structure. Mouse and human ZP genes are encoded by single-copy genes located on different chromosomes and are highly expressed in the ovary by growing oocytes during late stages of oogenesis. Genes encoding ZP proteins are conserved among mammals, and their expression is regulated by cis-acting sequences located close to the transcription start-site and by the same/similar trans-acting factors. Nascent ZP proteins are synthesized, packaged into vesicles, secreted into the extracellular space, and assembled into long, crosslinked fibrils that have a structural repeat, a ZP2-ZP3 dimer, and constitute the ZP matrix. Fibrils are oriented differently with respect to the oolemma in the inner and outer layers of the ZP. Sequence elements in the ZPD and the carboxy-terminal propeptide of ZP1–4 regulate secretion and assembly of nascent ZP proteins. The presence of both ZP2 and ZP3 is required to assemble ZP fibrils and ZP1 and ZP4 are used to crosslink the fibrils. Inactivation of mouse ZP genes by gene targeting has a detrimental effect on ZP formation around growing oocytes and female fertility. Gene sequence variations in human ZP genes due to point, missense, or frameshift mutations also have a detrimental effect on ZP formation and female fertility. The latter mutations provide additional support for the role of ZPD subdomains and other regions of ZP polypeptide in polymerization of human ZP proteins into fibrils and matrix.

Modulating the bicoid gradient in space and time

BackgroundThe formation of the Bicoid (Bcd) gradient in the early Drosophila is one of the most fascinating observations in biology and serves as a paradigm for gradient formation, yet its mechanism is still not fully understood. Two distinct models were proposed in the past, the SDD and the ARTS model.ResultsWe define novel cis- and trans-acting factors that are indispensable for gradient formation. The first one is the poly A tail length of the bcd mRNA where we demonstrate that it changes not only in time, but also in space. We show that posterior bcd mRNAs possess a longer poly tail than anterior ones and this elongation is likely mediated by wispy (wisp), a poly A polymerase. Consequently, modulating the activity of Wisp results in changes of the Bcd gradient, in controlling downstream targets such as the gap and pair-rule genes, and also in influencing the cuticular pattern. Attempts to modulate the Bcd gradient by subjecting the egg to an extra nuclear cycle, i.e. a 15th nuclear cycle by means of the maternal haploid (mh) mutation showed no effect, neither on the appearance of the gradient nor on the control of downstream target. This suggests that the segmental anlagen are determined during the first 14 nuclear cycles. Finally, we identify the Cyclin B (CycB) gene as a trans-acting factor that modulates the movement of Bcd such that Bcd movement is allowed to move through the interior of the egg.ConclusionsOur analysis demonstrates that Bcd gradient formation is far more complex than previously thought requiring a revision of the models of how the gradient is formed.

SDE2 is an essential gene required for ribosome biogenesis and the regulation of alternative splicing.

RNA provides the framework for the assembly of some of the most intricate macromolecular complexes within the cell, including the spliceosome and the mature ribosome. The assembly of these complexes relies on the coordinated association of RNA with hundreds of trans-acting protein factors. While some of these trans-acting factors are RNA-binding proteins (RBPs), others are adaptor proteins, and others still, function as both. Defects in the assembly of these complexes results in a number of human pathologies including neurodegeneration and cancer. Here, we demonstrate that Silencing Defective 2 (SDE2) is both an RNA binding protein and also a trans-acting adaptor protein that functions to regulate RNA splicing and ribosome biogenesis. SDE2 depletion leads to widespread changes in alternative splicing, defects in ribosome biogenesis and ultimately complete loss of cell viability. Our data highlight SDE2 as a previously uncharacterized essential gene required for the assembly and maturation of the complexes that carry out two of the most fundamental processes in mammalian cells.

Annexin A2 binds the internal ribosomal entry site of c-myc mRNA and regulates its translation

ABSTRACT The expression and localization of the oncoprotein c-Myc is highly regulated at the level of transcription, mRNA transport, translation, as well as stability of the protein. We previously showed that Annexin A2 (AnxA2) binds to a specific localization element in the 3ʹuntranslated region (UTR) of c-myc mRNA and is involved in its localization to the perinuclear region. In the present study, we demonstrate that AnxA2 binds in a Ca 2+ -dependent manner to the internal ribosomal entry site (IRES) containing two pseudo-knots in the 5´UTR of the c-myc mRNA. Here, we employ an in vitro rabbit reticulocyte lysate system with chimeric c-myc reporter mRNAs to demonstrate that binding of AnxA2 to the c-myc IRES modulates the expression of c-Myc. Notably, we show that low levels of AnxA2 appear to increase, while high levels of AnxA2 inhibits translation of the chimeric mRNA. However, when both the AnxA2-binding site and the ribosomal docking site in the c-myc IRES are deleted, AnxA2 has no effect on the translation of the reporter mRNA. Forskolin-treatment of PC12 cells results in upregulation of Ser25 phosphorylated AnxA2 expression while c-Myc expression is down-regulated. The effect of forskolin on c-Myc expression and the level of Ser25 phosphorylated AnxA2 was abolished in the presence of EGTA. These findings indicate that AnxA2 regulates both the transport and subsequent translation of the c-myc mRNA, possibly by silencing the mRNA during its transport. They also suggest that AnxA2 act as a switch to turn off the c-myc IRES activity in the presence of calcium. Abbreviations: AnxA2, Annexin A2; β2--µglob, β2-microglobulin; cpm, counts per minute; hnRNP, heterogenous nuclear ribonucleoprotein; IRES, internal ribosomal entry site; ITAF, IRES trans-acting factor; MM, multiple myeloma; PABP, poly(A)-binding protein; PCBP, poly(rC) binding protein; PSF, PTB-associated splicing factor; PTB, polypyrimidine tract binding protein; RRL, rabbit reticulocyte lysate; UTR, untranslated region; YB, Y-box binding protein.

Variants in a cis-regulatory element of TBX1 in conotruncal heart defect patients impair GATA6-mediated transactivation

BackgroundTBX1 (T-box transcription factor 1) is a major candidate gene that likely contributes to the etiology of velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS). Although the haploinsufficiency of TBX1 in both mice and humans results in congenital cardiac malformations, little has been elucidated about its upstream regulation. We aimed to explore the transcriptional regulation and dysregulation of TBX1.MethodsDifferent TBX1 promoter reporters were constructed. Luciferase assays and electrophoretic mobility shift assays (EMSAs) were used to identify a cis-regulatory element within the TBX1 promoter region and its trans-acting factor. The expression of proteins was identified by immunohistochemistry and immunofluorescence. Variants in the cis-regulatory element were screened in conotruncal defect (CTD) patients. In vitro functional assays were performed to show the effects of the variants found in CTD patients on the transactivation of TBX1.ResultsWe identified a cis-regulatory element within intron 1 of TBX1 that was found to be responsive to GATA6 (GATA binding protein 6), a transcription factor crucial for cardiogenesis. The expression patterns of GATA6 and TBX1 overlapped in the pharyngeal arches of human embryos. Transfection experiments and EMSA indicated that GATA6 could activate the transcription of TBX1 by directly binding with its GATA cis-regulatory element in vitro. Furthermore, sequencing analyses of 195 sporadic CTD patients without the 22q11.2 deletion or duplication identified 3 variants (NC_000022.11:g.19756832C > G, NC_000022.11:g.19756845C > T, and NC_000022.11:g. 19756902G > T) in the non-coding cis-regulatory element of TBX1. Luciferase assays showed that all 3 variants led to reduced transcription of TBX1 when incubated with GATA6.ConclusionsOur findings showed that TBX1 might be a direct transcriptional target of GATA6, and variants in the non-coding cis-regulatory element of TBX1 disrupted GATA6-mediated transactivation.

3D or Not 3D: Shaping the Genome during Development.

One of the most fundamental questions in developmental biology is how one fertilized cell can give rise to a fully mature organism and how gene regulation governs this process. Precise spatiotemporal gene expression is required for development and is believed to be achieved through a complex interplay of sequence-specific information, epigenetic modifications, trans-acting factors, and chromatin folding. Here we review the role of chromatin folding during development, the mechanisms governing 3D genome organization, and how it is established in the embryo. Furthermore, we discuss recent advances and debated questions regarding the contribution of the 3D genome to gene regulation during organogenesis. Finally, we describe the mechanisms that can reshape the 3D genome, including disease-causing structural variations and the emerging view that transposable elements contribute to chromatin organization.

Molecular characterization of a putative new cavemovirus isolated from wild chicory (Cichorium intybus).

A putative new virus with sequence similarity to members of the genus Cavemovirus in the family Caulimoviridae was identified in wild chicory (Cichorium intybus) by next-generation sequencing (NGS). The putative new virus was tentatively named "chicory mosaic cavemovirus" (ChiMV), and its genome was determined to be 7,775 nucleotides (nt) long with the typical genome organization of cavemoviruses. ORF1 encodes a putative coat protein/movement polyprotein (1,278 aa), ORF2 encodes a putative replicase (650 aa), and ORF3 encodes a putative transactivator factor (384 aa). The first two putative proteins have 46.2% and 68.7% amino acid sequence identity to the CP/MP protein (YP_004347414) and replicase (YP_004347415), respectively, of sweet potato collusive virus (SPCV). ORF3 encodes a protein with 38.5% amino acid sequence identity to the putative transactivator factor (NP_056849) of cassava vein mosaic virus (CsVMV). The new putative viral genome and those of three cavemoviruses (epiphyllum virus 4 [EpV-4], SPCV, and CsVMV) differ by 24-27% in the nt sequence of the replicase gene, which exceeds the species demarcation cutoff (>20%) for the family.

Genetic architecture modulates diet-induced hepatic mRNA and miRNA expression profiles in Diversity Outbred mice.

Genetic approaches in model organisms have consistently demonstrated that molecular traits such as gene expression are under genetic regulation, similar to clinical traits. The resulting expression quantitative trait loci (eQTL) have revolutionized our understanding of genetic regulation and identified numerous candidate genes for clinically relevant traits. More recently, these analyses have been extended to other molecular traits such as protein abundance, metabolite levels, and miRNA expression. Here, we performed global hepatic eQTL and microRNA expression quantitative trait loci (mirQTL) analysis in a population of Diversity Outbred mice fed two different diets. We identified several key features of eQTL and mirQTL, namely differences in the mode of genetic regulation (cis or trans) between mRNA and miRNA. Approximately 50% of mirQTL are regulated by a trans-acting factor, compared to ∼25% of eQTL. We note differences in the heritability of mRNA and miRNA expression and variance explained by each eQTL or mirQTL. In general, cis-acting variants affecting mRNA or miRNA expression explain more phenotypic variance than trans-acting variants. Finally, we investigated the effect of diet on the genetic architecture of eQTL and mirQTL, highlighting the critical effects of environment on both eQTL and mirQTL. Overall, these data underscore the complex genetic regulation of two well-characterized RNA classes (mRNA and miRNA) that have critical roles in the regulation of clinical traits and disease susceptibility

QuantifyPoly(A): reshaping alternative polyadenylation landscapes of eukaryotes with weighted density peak clustering.

The dynamic choice of different polyadenylation sites in a gene is referred to as alternative polyadenylation, which functions in many important biological processes. Large-scale messenger RNA 3' end sequencing has revealed that cleavage sites for polyadenylation are presented with microheterogeneity. To date, the conventional determination of polyadenylation site clusters is subjective and arbitrary, leading to inaccurate annotations. Here, we present a weighted density peak clustering method, QuantifyPoly(A), to accurately quantify genome-wide polyadenylation choices. Applying QuantifyPoly(A) on published 3' end sequencing datasets from both animals and plants, their polyadenylation profiles are reshaped into myriads of novel polyadenylation site clusters. Most of these novel polyadenylation site clusters show significantly dynamic usage across different biological samples or associate with binding sites of trans-acting factors. Upstream sequences of these clusters are enriched with polyadenylation signals UGUA, UAAA and/or AAUAAA in a species-dependent manner. Polyadenylation site clusters also exhibit species specificity, while plants ones generally show higher microheterogeneity than that of animals. QuantifyPoly(A) is broadly applicable to any types of 3' end sequencing data and species for accurate quantification and construction of the complex and dynamic polyadenylation landscape and enables us to decode alternative polyadenylation events invisible to conventional methods at a much higher resolution.

Emotions in the Electronic Educational Space

The study of the role of emotions in education has already established traditions. The relevance of studying emotions in the electronic educational space stems from the expansion of distance education both in Russia and in other countries. System analysis allows us to identify many functions that emotions perform in learning, which include: motivational, meaning-formative, regulatory, communicative, directing, activating, the function of increasing creative potential, integrative, socializing, goal-setting. The retransmission of emotions in the electronic educational space has its own characteristics. Text information plays an important role in e-education. Therefore, the linguistics of emotions, discursive emotions and forms of verbalization of emotiongenic information are of great importance. The concepts of “emotional design” are of methodological importance for the study of emotions in the electronic educational space. The study revealed the different significance of cognitive and transactional emotiongenic factors of electronic space design for students. The most important cognitive emotiongenic factors were named: brightness and accessibility of visual signs, traditions and “customs”, feedback, reliability of the electronic system. The most significant transactional emotiongenic factors were: social communication, visualization, individualization in the design of the site, retransmission of values through the electronic educational space. The significance of emotiongenic factors differs on ZOOM, MOODLE, and LMS. The study of emotions when working in the electronic space allows us to identify areas of University information educational environment development.

Translation Initiation Regulated by RNA-Binding Protein in Mammals: The Modulation of Translation Initiation Complex by Trans-Acting Factors.

Protein synthesis is tightly regulated at each step of translation. In particular, the formation of the basic cap-binding complex, eukaryotic initiation factor 4F (eIF4F) complex, on the 5′ cap structure of mRNA is positioned as the rate-limiting step, and various cis-elements on mRNA contribute to fine-tune spatiotemporal protein expression. The cis-element on mRNAs is recognized and bound to the trans-acting factors, which enable the regulation of the translation rate or mRNA stability. In this review, we focus on the molecular mechanism of how the assembly of the eIF4F complex is regulated on the cap structure of mRNAs. We also summarize the fine-tuned regulation of translation initiation by various trans-acting factors through cis-elements on mRNAs.

A ratiometric dual luciferase reporter for quantitative monitoring of pre-mRNA splicing efficiency in vivo

Precursor messenger RNA (pre-mRNA) splicing is critical for cell growth and development, and errors in RNA splicing frequently cause cellular dysfunction, abnormal gene expression, and a variety of human diseases. However, there is currently a lack of reliable systems to noninvasively monitor the mRNA splicing efficiency in cells and animals. Here, we described the design of a genetically engineered ratiometric dual luciferase reporter to continuously quantify the changes in mRNA splice variants in vivo. This reporter system is encoded within a single polypeptide but on separate exons, thus generating two distinct luciferase signals derived from spliced and unspliced mRNAs. With this reporter, the two kinds of luciferase in the same individual can minimize the influence of indirect factors on splicing, and the ratio of these two luciferase intensities represents the dynamic splicing efficiency of pre-mRNA. Our study offers a convenient and robust tool for the screening and identification of small molecules or trans-acting factors that affect the efficiency of specific splicing reactions.

The Causes and Consequences of Spatial Organization of the Genome in Regulation of Gene Expression.

Regulation of gene expression in time, space and quantity is orchestrated by the functional interplay of cis-acting elements and trans-acting factors. Our current view postulates that transcription factors recognize enhancer DNA and read the transcriptional regulatory code by cooperative DNA binding to specific DNA motifs, thus instructing the recruitment of transcriptional regulatory complexes forming a plethora of higher-ordered multi-protein-DNA and protein-protein complexes. Here, we reviewed the formation of multi-dimensional chromatin assemblies implicated in gene expression with emphasis on the regulatory role of enhancer hubs as coordinators of stochastic gene expression. Enhancer hubs contain many interacting regulatory elements and represent a remarkably dynamic and heterogeneous network of multivalent interactions. A functional consequence of such complex interaction networks could be that individual enhancers function synergistically to ensure coordination, tight control and robustness in regulation of expression of spatially connected genes. In this review, we discuss fundamental paradigms of such inter- and intra- chromosomal associations both in the context of immune-related genes and beyond.

APOBEC1 mediated C-to-U RNA editing: target sequence and trans-acting factor contribution to 177 RNA editing events in 119 murine transcripts in-vivo.

Mammalian C-to-U RNA editing was described more than 30 yr ago as a single nucleotide modification in small intestinal Apob RNA, later shown to be mediated by the RNA-specific cytidine deaminase APOBEC1. Reports of other examples of C-to-U RNA editing, coupled with the advent of genome-wide transcriptome sequencing, identified an expanded range of APOBEC1 targets. Here we analyze the cis-acting regulatory components of verified murine C-to-U RNA editing targets, including nearest neighbor as well as flanking sequence requirements and folding predictions. RNA secondary structure of the editing cassette was associated with editing frequency and exhibited minimal free energy values comparable to small nuclear RNAs. We summarize findings demonstrating the relative importance of trans-acting factors (A1CF, RBM47) acting in concert with APOBEC1. Cofactor dominance was associated with editing frequency, with RNAs targeted by both RBM47 and A1CF edited at a lower frequency than RBM47-dominant targets. Using this information, we developed a multivariable linear regression model to predict APOBEC1 dependent C-to-U RNA editing efficiency, incorporating factors independently associated with editing frequencies based on 103 Sanger-confirmed editing sites, which accounted for 84% of the observed variance. This model also predicted a composite score for available human C-to-U RNA targets, which again correlated with editing frequency.