Putative RNA-binding Protein Research Articles

Comparative Genomics and Evolutionary Analysis of RNA-Binding Proteins of Burkholderia cenocepacia J2315 and Other Members of the B. cepacia Complex.

RNA-binding proteins (RBPs) are important regulators of cellular functions, playing critical roles on the survival of bacteria and in the case of pathogens, on their interaction with the host. RBPs are involved in transcriptional, post-transcriptional, and translational processes. However, except for model organisms like Escherichia coli, there is little information about the identification or characterization of RBPs in other bacteria, namely in members of the Burkholderia cepacia complex (Bcc). Bcc is a group of bacterial species associated with a poor clinical prognosis in cystic fibrosis patients. These species have some of the largest bacterial genomes, and except for the presence of two-distinct Hfq-like proteins, their RBP repertoire has not been analyzed so far. Using in silico approaches, we identified 186 conventional putative RBPs in Burkholderia cenocepacia J2315, an epidemic and multidrug resistant pathogen of cystic fibrosis patients. Here we describe the comparative genomics and phylogenetic analysis of RBPs present in multiple copies and predicted to play a role in transcription, protein synthesis, and RNA decay in Bcc bacteria. In addition to the two different Hfq chaperones, five cold shock proteins phylogenetically close to E. coli CspD protein and three distinct RhlE-like helicases could be found in the B. cenocepacia J2315 genome. No RhlB, SrmB, or DeaD helicases could be found in the genomes of these bacteria. These results, together with the multiple copies of other proteins generally involved in RNA degradation, suggest the existence, in B. cenocepacia and in other Bcc bacteria, of some extra and unexplored functions for the mentioned RBPs, as well as of alternative mechanisms involved in RNA regulation and metabolism in these bacteria.

Interactive Alternative Splicing Analysis of Human Stem Cells Using psichomics.

Alternative splicing (AS) generates functionally distinct transcripts and is involved in multiple cellular processes, including stem cell differentiation. Several epithelial-to-mesenchymal transition-related splicing factors have also been associated with pluripotency. Concomitantly with the interest in studying AS in stem cell biology, the advent of next-generation sequencing of RNA (RNA-seq) has increased the public availability of transcriptomic data and enabled genome-wide AS studies. To facilitate performing such analyses in large publicly available or user-provided transcriptomics datasets, the psichomics R package provides an easy-to-use interface and efficient data visualization tools for AS quantification and integrative analyses of AS and gene expression data.psichomics is employed herein to study AS changes between human stem cells and fibroblasts, based on dimensionality reduction, and median- and variance-based differential AS and gene expression analyses. Putative RNA-binding protein regulators involved in those alterations are then identified based on correlation analyses in large cohorts of human tissue transcriptomes. We identified several alterations, both novel and previously reported, in alternative splicing events and in the expression of their candidate regulators that are associated with stem cell differentiation into fibroblasts.

ORNAment: a database of putative RNA binding protein target sites in the transcriptomes of model species.

Protein–RNA interactions are essential for controlling most aspects of RNA metabolism, including synthesis, processing, trafficking, stability and degradation. In vitro selection methods, such as RNAcompete and RNA Bind-n-Seq, have defined the consensus target motifs of hundreds of RNA-binding proteins (RBPs). However, readily available information about the distribution features of these motifs across full transcriptomes was hitherto lacking. Here, we introduce oRNAment (o RNA motifs enrichment in transcriptomes), a database that catalogues the putative motif instances of 223 RBPs, encompassing 453 motifs, in a transcriptome-wide fashion. The database covers 525 718 complete coding and non-coding RNA species across the transcriptomes of human and four prominent model organisms: Caenorhabditis elegans, Danio rerio, Drosophila melanogaster and Mus musculus. The unique features of oRNAment include: (i) hosting of the most comprehensive mapping of RBP motif instances to date, with 421 133 612 putative binding sites described across five species; (ii) options for the user to filter the data according to a specific threshold; (iii) a user-friendly interface and efficient back-end allowing the rapid querying of the data through multiple angles (i.e. transcript, RBP, or sequence attributes) and (iv) generation of several interactive data visualization charts describing the results of user queries. oRNAment is freely available at http://rnabiology.ircm.qc.ca/oRNAment/.

Sequestration of microRNA-mediated target repression by the Ago2-associated RNA-binding protein FAM120A.

Argonaute (Ago) proteins interact with various binding partners and play a pivotal role in microRNA (miRNA)-mediated silencing pathways. By utilizing immunoprecipitation followed by mass spectrometry to determine cytoplasmic Ago2 protein complexes in mouse embryonic stem cells (mESCs), we identified a putative RNA-binding protein FAM120A (also known as OSSA/C9ORF10) as an Ago2 interacting protein. Individual nucleotide resolution cross-linking and immunoprecipitation (iCLIP) analysis revealed that FAM120A binds to homopolymeric tracts in 3′-UTRs of about 2000 mRNAs, particularly poly(G) sequences. Comparison of FAM120A iCLIP and Ago2 iCLIP reveals that greater than one-third of mRNAs bound by Ago2 in mESCs are co-bound by FAM120A. Furthermore, such FAM120A-bound Ago2 target genes are not subject to Ago2-mediated target degradation. Reporter assays suggest that the 3′-UTRs of several FAM120A-bound miRNA target genes are less sensitive to Ago2-mediated target repression than those of FAM120A-unbound miRNA targets and FAM120A modulates them via its G-rich target sites. These findings suggest that Ago2 may exist in multiple protein complexes with varying degrees of functionality.

PTB-AS, a Novel Natural Antisense Transcript, Promotes Glioma Progression by Improving PTBP1 mRNA Stability with SND1.

Glioma, the most common primary malignancy in the brain, has high recurrence and lethality rates, and thus, elucidation of the molecular mechanisms of this incurable disease is urgently needed. Poly-pyrimidine tract binding protein (PTBP1, also known as hnRNP I), an RNA-binding protein, has various mechanisms to promote gliomagenesis. However, the mechanisms regulating PTBP1 expression are unclear. Herein, we report a novel natural antisense noncoding RNA, PTB-AS, whose expression correlated positively with PTBP1 mRNA. We found that PTB-AS significantly promoted the proliferation and migration invivo and invitro of glioma cells. PTB-AS substantially increased the PTBP1 level by directly binding to its 3' UTR and stabilizing the mRNA. Furthermore, staphylococcal nuclease domain-containing 1 (SND1) dramatically increased the binding capacity between PTB-AS and PTBP1 mRNA. Mechanistically, PTB-AS could mask the binding site of miR-9 in the PTBP1-3' UTR; miR-9 negatively regulates PTBP1. To summarize, we revealed that PTB-AS, which maintains the PTBP1 level through extended base pairing to the PTBP1 3' UTR with the assistance of SND1, could significantly promote gliomagenesis.

Achilles-Mediated and Sex-Specific Regulation of Circadian mRNA Rhythms in Drosophila.

The circadian clock is an evolutionarily conserved mechanism that generates the rhythmic expression of downstream genes. The core circadian clock drives the expression of clock-controlled genes, which in turn play critical roles in carrying out many rhythmic physiological processes. Nevertheless, the molecular mechanisms by which clock output genes orchestrate rhythmic signals from the brain to peripheral tissues are largely unknown. Here we explored the role of one rhythmic gene, Achilles, in regulating the rhythmic transcriptome in the fly head. Achilles is a clock-controlled gene in Drosophila that encodes a putative RNA-binding protein. Achilles expression is found in neurons throughout the fly brain using fluorescence in situ hybridization (FISH), and legacy data suggest it is not expressed in core clock neurons. Together, these observations argue against a role for Achilles in regulating the core clock. To assess its impact on circadian mRNA rhythms, we performed RNA sequencing (RNAseq) to compare the rhythmic transcriptomes of control flies and those with diminished Achilles expression in all neurons. Consistent with previous studies, we observe dramatic upregulation of immune response genes upon knock-down of Achilles. Furthermore, many circadian mRNAs lose their rhythmicity in Achilles knock-down flies, suggesting that a subset of the rhythmic transcriptome is regulated either directly or indirectly by Achilles. These Achilles-mediated rhythms are observed in genes involved in immune function and in neuronal signaling, including Prosap, Nemy and Jhl-21. A comparison of RNAseq data from control flies reveals that only 42.7% of clock-controlled genes in the fly brain are rhythmic in both males and females. As mRNA rhythms of core clock genes are largely invariant between the sexes, this observation suggests that sex-specific mechanisms are an important, and heretofore under-appreciated, regulator of the rhythmic transcriptome.

NS38 is required for aquareovirus replication via interaction with viral core proteins and host eIF3A

Aquareoviruses contain an 11-segmented double-stranded RNA genome. Previous studies indicated that NS38, a virus-encoded putative single-stranded RNA binding protein, interacts with NS80 in viral inclusion bodies (VIBs). However, the role of NS38 in aquareovirus infection remained unclear. Here, we found that NS38 interacts with inner-capsid proteins (VP1–VP4 and VP6) and the NS80-RNA complex in both transfected and infected cells. Knockdown of NS38 by siRNAs-115/219 clearly reduced viral infection, with decreased mRNA and protein yields. Moreover, NS38 can interact with host cellular eukaryotic translation initiation factor 3 subunit A (eIF3A) in transfected cells, while no association was detected between eIF3A and NS80. This study is the first to define that the NS38 is essential to viral replication. Together, our findings indicate that NS38 might function as a mediator by interacting with viral and host cellular components in VIBs during replication.

LARGE GRAIN Encodes a Putative RNA-Binding Protein that Regulates Spikelet Hull Length in Rice

Grain size is a key determiner of grain weight, one of the yield components in rice (Oryza sativa). Therefore, to increase grain yield, it is important to elucidate the detailed mechanisms regulating grain size. The Large grain (Lgg) mutant, found in the nonautonomous DNA-based active rice transposon1 (nDart1)-tagged lines of Koshihikari, is caused by a truncated nDart1-3 and 355 bp deletion in the 5' untranslated region of LGG, which encodes a putative RNA-binding protein, through transposon display and cosegregation analysis between grain length and LGG genotype in F2 and F3. Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9-mediated knockout and overexpression of LGG led to longer and shorter grains than wild type, respectively, showing that LGG regulates spikelet hull length. Expression of LGG was highest in the 0.6-mm-long young panicle and gradually decreased as the panicle elongated. LGG was also expressed in roots and leaves. These results show that LGG functions at the very early stage of panicle development. Longitudinal cell numbers of spikelet hulls of Lgg, knockout and overexpressed plants were significantly different from those of the wild type, suggesting that LGG might regulate longitudinal cell proliferation in the spikelet hull. RNA-Seq analysis of 1-mm-long young panicles from LGG knockout and overexpressing plants revealed that the expressions of many cell cycle-related genes were reduced in knockout plants relative to LGG-overexpressing plants and wild type, whereas some genes for cell proliferation were highly expressed in knockout plants. Taken together, these results suggest that LGG might be a regulator of cell cycle and cell division in the rice spikelet hull.

Macromolecular Connectivity Landscape of Mammalian Brain

Connectivity webs mediate the unique biology of the mammalian brain. Yet while cell circuit maps are increasingly available, knowledge of the underlying molecular networks remains limited. Here, we applied multi-dimensional biochemical fractionation with precision mass spectrometry and machine learning to survey endogenous macromolecules in adult mouse brain. We defined a global ‘interactome’ of multi-protein complexes, most never reported before. These brain-selective assemblies have distinct physical and functional attributes and show regional- and cell-type specificity. A striking number are also linked to neurological disorders and disease variants with broad pathophysiological relevance. Using reciprocal pulldowns and a transgenic model, we validated a putative 15-member RNA-binding protein complex associated with amyotrophic lateral sclerosis, establishing regulatory functions in alternative splicing and disease progression. This Brain Interaction Map – or BraInMap – resource facilitates mechanistic exploration of the molecular machinery driving core processes and diseases of the central nervous system.

A real-time PCR assay for quantification of parasite burden in murine models of leishmaniasis.

Eukaryotic parasites in the genus Leishmania place approximately 350 million people per year at risk of disease. In addition to their global health significance, Leishmania spp. have served as an important model for delineating basic concepts in immunology such as T-helper cell polarization. There have been many qPCR-based assays reported for measuring parasite burden in humans and animals. However, these are largely optimized for use in clinical diagnosis and not specifically for animal models. This has led several of these assays to have suboptimal characteristics for use in animal models. For example, multi-copy number genes have been frequently used to increase sensitivity but are subject to greater plasticity within the genome and thus may confound effects of experimental manipulations in animal models. In this study, we developed a sybr-green based quantitative touchdown PCR assay for a highly conserved and single-copy putative RNA-binding protein, DRBD3. With primers that share greater than 90% sequence identity across all sequenced Leishmania spp., we demonstrate that this assay has a lower limit of detection of 100 fg of parasite DNA for Leishmania major, L. donovani, L. venezuelensis, and L. panamensis. Using C57BL6/J mice, we used this assay to monitor parasite burden over 1 month of infection with two strains of L. major (Seidman and Friedlin), and L. venezeuelensis. These characteristics rival the sensitivity of previously reported qPCR based methods of parasite quantitation while amplifying a stable, single copy gene. Use of this protocol in the future will lead to improved accuracy in animal based models and help to tease apart differences in biology of host-parasite interactions.

A Chemical Biology Approach to Model Pontocerebellar Hypoplasia Type 1B (PCH1B).

Mutations of EXOSC3 have been linked to the rare neurological disorder known as Pontocerebellar Hypoplasia type 1B (PCH1B). EXOSC3 is one of three putative RNA-binding structural cap proteins that guide RNA into the RNA exosome, the cellular machinery that degrades RNA. Using RNAcompete, we identified a G-rich RNA motif binding to EXOSC3. Surface plasmon resonance (SPR) and microscale thermophoresis (MST) indicated an affinity in the low micromolar range of EXOSC3 for long and short G-rich RNA sequences. Although several PCH1B-causing mutations in EXOSC3 did not engage a specific RNA motif as shown by RNAcompete, they exhibited lower binding affinity to G-rich RNA as demonstrated by MST. To test the hypothesis that modification of the RNA-protein interface in EXOSC3 mutants may be phenocopied by small molecules, we performed an in-silico screen of 50 000 small molecules and used enzyme-linked immunosorbant assays (ELISAs) and MST to assess the ability of the molecules to inhibit RNA-binding by EXOSC3. We identified a small molecule, EXOSC3-RNA disrupting (ERD) compound 3 (ERD03), which ( i) bound specifically to EXOSC3 in saturation transfer difference nuclear magnetic resonance (STD-NMR), ( ii) disrupted the EXOSC3-RNA interaction in a concentration-dependent manner, and ( iii) produced a PCH1B-like phenotype with a 50% reduction in the cerebellum and an abnormally curved spine in zebrafish embryos. This compound also induced modification of zebrafish RNA expression levels similar to that observed with a morpholino against EXOSC3. To our knowledge, this is the first example of a small molecule obtained by rational design that models the abnormal developmental effects of a neurodegenerative disease in a whole organism.

In Silico Prediction and Validation of Novel RNA Binding Proteins and Residues in the Human Proteome.

Deciphering a complete landscape of protein-RNA interactions in the human proteome remains an elusive challenge. We computationally elucidate RNA binding proteins (RBPs) using an approach that complements previous efforts. We employ two modern complementary sequence-based methods that provide accurate predictions from the structured and the intrinsically disordered sequences, even in the absence of sequence similarity to the known RBPs. We generate and analyze putative RNA binding residues on the whole proteome scale. Using a conservative setting that ensures low, 5% false positive rate, we identify 1511 putative RBPs that include 281 known RBPs and 166 RBPs that were previously predicted. We empirically demonstrate that these overlaps are statistically significant. We also validate the putative RBPs based on two major hallmarks of their RNA binding residues: high levels of evolutionary conservation and enrichment in charged amino acids. Moreover, we show that the novel RBPs are significantly under-annotated functionally which coincides with the fact that they were not yet found to interact with RNAs. We provide two examples of our novel putative RBPs for which there is recent evidence of their interactions with RNAs. The dataset of novel putative RBPs and RNA binding residues for the future hypothesis generation is provided in the Supporting Information.

A second RNA-binding protein is essential for ethanol tolerance provided by the bacterial OLE ribonucleoprotein complex

OLE (ornate, large, extremophilic) RNAs comprise a class of structured noncoding RNAs (ncRNAs) found in many extremophilic bacteria species. OLE RNAs constitute one of the longest and most widespread bacterial ncRNA classes whose major biochemical function remains unknown. In the Gram-positive alkaliphile Bacillus halodurans, OLE RNA is abundant, and localizes to the cell membrane by association with the transmembrane OLE-associated protein called OapA (formerly OAP). These characteristics, along with the well-conserved sequence and structural features of OLE RNAs, suggest that the OLE ribonucleoprotein (RNP) complex performs important biological functions. B. halodurans strains lacking OLE RNA (∆ole) or OapA (∆oapA) are less tolerant of cold (20 °C) and short-chain alcohols (e.g., ethanol). Here, we describe the effects of a mutant OapA (called PM1) that more strongly inhibits growth under cold or ethanol stress compared with strains lacking the oapA gene, even when wild-type OapA is present. This dominant-negative effect of PM1 is reversed by mutations that render OLE RNA nonfunctional. This finding demonstrates that the deleterious PM1 phenotype requires an intact RNP complex, and suggests that the complex has one or more additional undiscovered components. A genetic screen uncovered PM1 phenotype suppressor mutations in the ybzG gene, which codes for a putative RNA-binding protein of unknown biological function. We observe that YbzG protein (also called OapB) selectively binds OLE RNA in vitro, whereas a mutant version of the protein is not observed to bind OLE RNA. Thus, YbzG/OapB is an important component of the functional OLE RNP complex in B. halodurans.

A putative RNA binding protein from Plasmodium vivax apicoplast

Malaria is caused by Apicomplexa protozoans from the Plasmodium genus entering the bloodstream of humans and animals through the bite of the female mosquitoes. The annotation of the Plasmodium vivax genome revealed a putative RNA binding protein (apiRBP) that was predicted to be trafficked into the apicoplast, a plastid organelle unique to Apicomplexa protozoans. Although a 3D structural model of the apiRBP corresponds to a noncanonical RNA recognition motif with an additional C‐terminal α‐helix (α3), preliminary protein production trials were nevertheless unsuccessful. Theoretical solvation analysis of the apiRBP model highlighted an exposed hydrophobic region clustering α3. Hence, we used a C‐terminal GFP‐fused chimera to stabilize the highly insoluble apiRBP and determined its ability to bind U‐rich stretches of RNA. The affinity of apiRBP toward such RNAs is highly dependent on ionic strength, suggesting that the apiRBP–RNA complex is driven by electrostatic interactions. Altogether, apiRBP represents an attractive tool for apicoplast transcriptional studies and for antimalarial drug design.

Enforcing Post-Transcriptional Circuitries to Achieve Human Hematopoietic Stem Cell Expansion

The balance between hematopoietic stem cell (HSC) differentiation and self-renewal is central to clinical regenerative paradigms. Unravelling the precise molecular mechanisms that govern HSC fate choices will thus have far reaching consequences for the development of effective therapies for hematopoietic and immunological disorders. There is an emerging recognition that beyond transcription, HSC homeostasis is subject to post-transcriptional control by RNA-binding proteins (RBPs) that ensure precise control of gene expression by modulating mRNA splicing, polyadenylation, localization, degradation or translation. RBPs can synchronously regulate the fates of operationally similar RNAs, in what have been termed RNA regulons. We have used a variety of functional approaches, in combination with unbiased genome- and proteome-scale, methods to define the tenets that govern this regulation and to determine key downstream circuitries of stem cell-regulating RBPs whose targeting could provide the basis for novel regenerative treatments. Through loss-of-function efforts, we have identified the RBP, MSI2, as a required factor for human HSC maintenance. By contrast, at supraphysiological levels, MSI2 has a profound positive effect on human HSC self-renewal decisions. Using a combination of global profiling, including mapping MSI2's targets through cross-linking immunoprecipitation (CLIP)-seq, we show that MSI2 achieves its ex vivo self-renewal-promoting effects by directing a co-ordinated post-transcriptional repression of key targets within the aryl hydrocarbon receptor (AHR) pathway. We are currently exploring the “rules” by which MSI2 influences its downstream effects on target RNAs and how it functions, in combination with other protein interactors, to instill a putative RBP regulatory code in HSCs. HSCs exhibit highly unique epigenomes, transcriptomes and proteomes and it is this distinctive molecular landscape that provides the canvas upon which MSI2, and indeed any other HSC-specific RBP exert their post-transcriptional influence over stem cell function. As such, to decipher the bona fide RNA networks that RBPs function upon in HSCs and to understand how they influence this network to enforce self-renewal, we are working towards performing systematic studies of RBP regulons in these cells specifically. In turn these approaches are elucidating a host of RBPs and post-transcriptional control mechanisms previously unappreciated for their role in HSC control. When modulated appropriately, we can successfully tailor these post-transcriptional regulons to enforce desired HSC outputs ex vivo. In summary, approaches to elucidate key HSC-regulatory RBPs and their protein and RNA interactomes provide valuable insights into a layer of HSC control previously not well understood, and one that can be capitalized on to achieve successful HSC expansion. DisclosuresNo relevant conflicts of interest to declare.

Disclosing 3' UTR cis-elements and putative partners involved in gene expression regulation in Leishmania spp.

To identify putative cis-elements involved in gene expression regulation in Leishmania, we previously conducted an in silico investigation to find conserved intercoding sequences (CICS) in the genomes of L. major, L. infantum, and L. braziliensis. Here, the CICS databank was explored to search for sequences that were present in the untranslated regions (UTRs) of groups of genes showing similar expression profiles during in vitro differentiation. Using a selectable marker as a reporter gene, flanked by either an intact 3' UTR or a UTR lacking the conserved element, the regulatory role of a CICS was confirmed. We observed that the pattern of modulation of the mRNA levels was altered in the absence of the CICS. We also identified putative CICS RNA-binding proteins. This study suggests that the publicly available CICS database is a useful tool for identifying regulatory cis-elements for Leishmania genes and suggests the existence of post-transcriptional regulons in Leishmania.

Silencing of AtRAP, a target gene of a bacteria-induced small RNA, triggers antibacterial defense responses through activation of LSU2 and down-regulation of GLK1.

Plants fine-tune their sophisticated immunity systems in response to pathogen infections. We previously showed that AtlsiRNA-1, a bacteria-induced plant endogenous small interfering RNA, silences the AtRAP gene, which encodes a putative RNA binding protein. In this study, we demonstrate that AtRAP functions as a negative regulator in plant immunity by characterizing molecular and biological responses of the knockout mutant and overexpression lines of AtRAP upon bacterial infection. AtRAP is localized in chloroplasts and physically interacts with Low Sulfur Upregulated 2 (LSU2), which positively regulates plant defense. Our results suggest that AtRAP negatively regulates defense responses by suppressing LSU2 through physical interaction. We also detected downregulation of the transcription factor GOLDEN2-LIKE 1 (GLK1) in atrap-1 using microarray analysis. The glk1 glk2 double mutant showed enhanced resistance to Pseudomonas syringae pv. tomato, which is consistent with a previous study showing enhanced resistance of a glk1 glk2 double mutant to Hyaloperonospora arabidopsidis. Taken together, our data suggest that silencing of AtRAP by AtlsiRNA-1 upon bacterial infection triggers defense responses through regulation of LSU2 and GLK1.

Virulence of Pseudomonas syringae pv. tomato DC3000 Is Influenced by the Catabolite Repression Control Protein Crc.

Pseudomonas syringae infects diverse plant species and is widely used as a model system in the study of effector function and the molecular basis of plant diseases. Although the relationship between bacterial metabolism, nutrient acquisition, and virulence has attracted increasing attention in bacterial pathology, it is largely unexplored in P. syringae. The Crc (catabolite repression control) protein is a putative RNA-binding protein that regulates carbon metabolism as well as a number of other factors in the pseudomonads. Here, we show that deletion of crc increased bacterial swarming motility and biofilm formation. The crc mutant showed reduced growth and symptoms in Arabidopsis and tomato when compared with the wild-type strain. We have evidence that the crc mutant shows delayed hypersensitive response (HR) when infiltrated into Nicotiana benthamiana and tobacco. Interestingly, the crc mutant was more susceptible to hydrogen peroxide, suggesting that, in planta, the mutant may be sensitive to reactive oxygen species generated during pathogen-associated molecular pattern-triggered immunity (PTI). Indeed, HR was further delayed when PTI-induced tissues were challenged with the crc mutant. The crc mutant did not elicit an altered PTI response in plants compared with the wild-type strain. We conclude that Crc plays an important role in growth and survival during infection.

APRICOT: an integrated computational pipeline for the sequence-based identification and characterization of RNA-binding proteins

RNA-binding proteins (RBPs) have been established as core components of several post-transcriptional gene regulation mechanisms. Experimental techniques such as cross-linking and co-immunoprecipitation have enabled the identification of RBPs, RNA-binding domains (RBDs) and their regulatory roles in the eukaryotic species such as human and yeast in large-scale. In contrast, our knowledge of the number and potential diversity of RBPs in bacteria is poorer due to the technical challenges associated with the existing global screening approaches. We introduce APRICOT, a computational pipeline for the sequence-based identification and characterization of proteins using RBDs known from experimental studies. The pipeline identifies functional motifs in protein sequences using position-specific scoring matrices and Hidden Markov Models of the functional domains and statistically scores them based on a series of sequence-based features. Subsequently, APRICOT identifies putative RBPs and characterizes them by several biological properties. Here we demonstrate the application and adaptability of the pipeline on large-scale protein sets, including the bacterial proteome of Escherichia coli. APRICOT showed better performance on various datasets compared to other existing tools for the sequence-based prediction of RBPs by achieving an average sensitivity and specificity of 0.90 and 0.91 respectively. The command-line tool and its documentation are available at https://pypi.python.org/pypi/bio-apricot.

Comparative testis proteome of cattleyak from different developmental stages

Cattleyak (hybrid of cattle and yak) exhibit higher capability in adaptability and production than cattle and yak, while the infertility of F1 males greatly restricts the effective utilization of this hybrid and little progress has been made on investigating the mechanisms of the cattleyak infertility. Cattleyak individuals at three development stages (10, 12 and 14-month old) were sampled in this work and the isobaric tag for relative and absolute quantification method was employed to identify differences between their testicular proteomes. The proteomic analysis identified 318 proteins differentially expressed with significance at 12-month stage and 327 at 14-month compared with 10-month stage, respectively. Compared with the testicular proteome from 10-month cattleyak, the gene ontology (GO) annotations of the differentially expressed proteins at 12 months did not indicate significant differences from those at 14 months, which confirmed the histological observation that germ cell reduction was more obvious and spermatogenic arrest may become more serious in 12-month-old cattleyak. On the other hand, 56 differentially expressed proteins were coexpressed at 12 and 14-month stage compared with 10-month stage, in which 32 proteins were upregulated and 24 downregulated. GO analysis revealed that most of the differently expressed proteins were involved in the molecular function of catalytic activity, transporter activity, oxidoreductase activity and protein binding. Further analysis indicated that the differently expressed proteins including testis-expressed protein 101 precursor, RNA-binding motif protein, X chromosome, putative RNA-binding protein 3, heparin-binding proteins, tudor domain-containing protein 1, glutathione S-transferases (GSTA2, GSTP1), heat shock-related 70 kDa protein 2, estradiol 17-β-dehydrogenase11, 2,4-dienoyl-CoA reductase and peroxiredoxin-2 were possibly associated with testis development and spermatogenesis, which could be selected as candidate proteins in future study to examine the mechanisms of cattleyak infertility.