Putative RNA-binding Protein Research Articles

Galectin-3 does not interact with RNA directly.

Galectin-3, well characterized as a glycan binding protein, has been identified as a putative RNA binding protein, possibly through participation in pre-mRNA maturation through interactions with splicosomes. Given recent developments with cell surface RNA biology, the putative dual-function nature of galectin-3 evokes a possible non-classical connection between glycobiology and RNA biology. However, with limited functional evidence of a direct RNA interaction, many molecular-level observations rely on affinity reagents and lack appropriate genetic controls. Thus, evidence of a direct interaction remains elusive. We demonstrate that antibodies raised to endogenous human galectin-3 can isolate RNA-protein crosslinks, but this activity remains insensitive to LGALS3 knock-out. Proteomic characterization of anti-galectin-3 IPs revealed enrichment of galectin-3, but high abundance of hnRNPA2B1, an abundant, well-characterized RNA-binding protein with weak homology to the N-terminal domain of galectin-3, in the isolate. Genetic ablation of HNRNPA2B1, but not LGALS3, eliminates the ability of the anti-galectin-3 antibodies to isolate RNA-protein crosslinks, implying either an indirect interaction or cross-reactivity. To address this, we introduced an epitope tag to the endogenous C-terminal locus of LGALS3. Isolation of the tagged galectin-3 failed to reveal any RNA-protein crosslinks. This result suggests that the galectin-3 does not directly interact with RNA and may be misidentified as an RNA-binding protein, at least in HeLa where the putative RNA associations were first identified. We encourage further investigation of this phenomenon employ gene deletions and, when possible, endogenous epitope tags to achieve the specificity required to evaluate potential interactions.

SeedTransNet: a directional translational network revealing regulatory patterns during seed maturation and germination.

Seed maturation is the developmental process that prepares the embryo for the desiccated waiting period before germination. It is associated with a series of physiological changes leading to the establishment of seed dormancy, seed longevity, and desiccation tolerance. We studied translational changes during seed maturation and observed a gradual reduction in global translation during seed maturation. Transcriptome and translatome profiling revealed specific reduction in the translation of thousands of genes. By including previously published data on germination and seedling establishment, a regulatory network based on polysome occupancy data was constructed: SeedTransNet. Network analysis predicted translational regulatory pathways involving hundreds of genes with distinct functions. The network identified specific transcript sequence features suggesting separate translational regulatory circuits. The network revealed several seed maturation-associated genes as central nodes, and this was confirmed by specific seed phenotypes of the respective mutants. One of the regulators identified, an AWPM19 family protein, PM19-Like1 (PM19L1), was shown to regulate seed dormancy and longevity. This putative RNA-binding protein also affects the translational regulation of its target mRNA, as identified by SeedTransNet. Our data show the usefulness of SeedTransNet in identifying regulatory pathways during seed phase transitions.

Roles and interactions of the specialized initiation factors EIF4E2, EIF4E5, and EIF4E6 in Trypanosoma brucei: EIF4E2 maintains the abundances of S-phase mRNAs.

Trypanosoma brucei has six versions of the cap-binding translation initiation factor EIF4E. We investigated the functions of EIF4E2, EIF4E3, EIF4E5, and EIF4E6 in bloodstream forms. We confirmed the protein associations previously found in procyclic forms and detected specific copurification of some RNA-binding proteins. Bloodstream forms lacking EIF4E5 grew normally and differentiated to replication-incompetent procyclic forms. Depletion of EIF4E6 inhibited bloodstream-form trypanosome growth and translation. EIF4E2 copurified only the putative RNA-binding protein SLBP2. Bloodstream forms lacking EIF4E2 multiplied slowly, had a low maximal cell density, and expressed the stumpy-form marker PAD1 but showed no evidence for enhanced stumpy-form signaling. EIF4E2 knock-out cells differentiated readily to replication-competent procyclic forms. EIF4E2 was strongly associated with a subset of mRNAs that are maximally abundant in the S-phase, and these all had decreased abundances in EIF4E2 knock-out cells. Three EIF4E2 target mRNAs are also bound and stabilized by the Pumilio domain protein PUF9. Yeast 2-hybrid results suggested that PUF9 interacts directly with SLBP2, but PUF9 was not detected in EIF4E2 pull-downs. We speculate that the EIF4E2-SLBP2 complex might interact with its target mRNAs, perhaps via PUF9, only early during G1/S, stabilizing the mRNAs in preparation for translation later in S-phase or in early G2.

The RNA-bound proteome of MRSA reveals post-transcriptional roles for helix-turn-helix DNA-binding and Rossmann-fold proteins

RNA-binding proteins play key roles in controlling gene expression in many organisms, but relatively few have been identified and characterised in detail in Gram-positive bacteria. Here, we globally analyse RNA-binding proteins in methicillin-resistant Staphylococcus aureus (MRSA) using two complementary biochemical approaches. We identify hundreds of putative RNA-binding proteins, many containing unconventional RNA-binding domains such as Rossmann-fold domains. Remarkably, more than half of the proteins containing helix-turn-helix (HTH) domains, which are frequently found in prokaryotic transcription factors, bind RNA in vivo. In particular, the CcpA transcription factor, a master regulator of carbon metabolism, uses its HTH domain to bind hundreds of RNAs near intrinsic transcription terminators in vivo. We propose that CcpA, besides acting as a transcription factor, post-transcriptionally regulates the stability of many RNAs.

Identification of RBMX as a splicing regulator in Parkinsonian mimetic induced alternative splicing of α-synuclein

α-Synuclein (α-syn) plays a precipitating role in Parkinson's disease (PD) due to its tendency to form oligomers and fibrils. The presence of smaller isoforms of α-syn was widely noticed in the affected brain regions of PD patients. 112-synuclein (112-syn) which lacks exon-5, possess enhanced aggregation propensity and forms intracellular inclusions. However, the factors responsible for the skipping of exon-5 are not completely understood. In this context, we aimed to identity the cis & trans-acting elements governing alternative splicing (AS) events by the Parkinsonian agent (MPP+) using minigene constructs. Minigene-I and -II were constructed by pruning the intron-4 and -5 regions respectively without altering the branch point adenosine to preserve splicing machinery. Also, chimeric minigenes were engineered by replacing either 5′ (Mini-III) or 3′ (Mini-IV) flanking intronic regions of exon-5 with other intronic regions (intron-3 and -2) that are not responsive to MPP+ induced splicing. While all the above minigenes exhibited MPP+-induced skipping of exon-5, Minigene-III did not generate the spliced product indicating that the 5′ flanking intronic region (316 bp) of exon-5 possess cis-acting elements responsible for oxidant-induced alternative splicing. RNA-Binding Protein Database (RBDP) analysis revealed the presence of four putative RNA binding proteins (RBPs), namely, RBMX, MBNL1, KHDRBS3 and SFRS1 that may bind to the 316 bp region of intron-4and their expression was substantially reduced following MPP+ treatment. Further, overexpression of RBMX mitigated MPP+-induced generation of 112-syn and also reduced intracellular α-syn aggregates. Overall, our study identified the pivotal role of the splicing regulator, RBMX, in the pathophysiology of PD.

A Putative Plasmodium RNA-Binding Protein Plays a Critical Role in Female Gamete Fertility and Parasite Transmission to the Mosquito Vector.

Plasmodium falciparum sexual stage gametocytes are critical for parasite transmission from the human host to the mosquito vector. Mature gametocytes generate fertile male (micro-) or female (macro-) gametes upon activation inside the mosquito midgut. While a number of parasite genes have been described that are critical for P. falciparum gametogenesis and fertility, no parasite gene has been shown to have a unique function in macrogametes. The genome of P. falciparum encodes numerous RNA-binding proteins. We identified a novel protein containing a putative RNA-binding domain, which we named Macrogamete-Contributed Factor Essential for Transmission (MaCFET). This protein is expressed in the asexual and sexual stages. Parasites that carry a deletion of MaCFET (Pfmacfet¯), developed normally as asexual stages, indicating that its function is not essential for the asexual proliferation of the parasite in vitro. Furthermore, Pfmacfet¯ male and female gametocytes developed normally and underwent activation to form microgametes and macrogametes. However, by utilizing genetic crosses, we demonstrate that Pfmacfet¯ parasites suffer a complete female-specific defect in successful fertilization. Therefore, PfMaCFET is a critical female-contributed factor for parasite transmission to the mosquito. Based on its putative RNA-binding properties, PfMaCFET might be in involved in the regulation of mRNAs that encode female-specific functions for fertilization or female-contributed factors needed post fertilization.

Integrated In Silico Analyses Identify PUF60 and SF3A3 as New Spliceosome-Related Breast Cancer RNA-Binding Proteins.

Simple SummaryGlobally, breast cancer (BC) is the most common cancer in women. Although numerous studies have attempted to address this worldwide health problem, it has not yet been possible to understand cancer in its entirety, mainly because most of the investigations have been focused on traditional molecular traits of DNA. Thus, new characteristics of breast tumorigenesis must be tackled, such as RNA-binding proteins (RBPs), which are crucial regulators of important cellular processes. To identify novel breast cancer RNA-binding proteins, we integrated several bioinformatic resources derived from experimentation on BC patient samples and cell lines. Consequently, we identified five putative breast cancer RNA-binding proteins (PUF60, TFRC, KPNB1, NSF, and SF3A3) showing strong tumorigenic characteristics. Supplementary investigation of the molecular and cellular functions of these proteins identified PUF60 and SF3A3 as new spliceosome-related breast cancer RNA-binding proteins. Further experimentation should center on these five RBPs to identify their role in breast tumorigenesis and potentially discover new druggable targets.More women are diagnosed with breast cancer (BC) than any other type of cancer. Although large-scale efforts have completely redefined cancer, a cure remains unattainable. In that respect, new molecular functions of the cell should be investigated, such as post-transcriptional regulation. RNA-binding proteins (RBPs) are emerging as critical post-transcriptional modulators of tumorigenesis, but only a few have clear roles in BC. To recognize new putative breast cancer RNA-binding proteins, we performed integrated in silico analyses of all human RBPs (n = 1392) in three major cancer databases and identified five putative BC RBPs (PUF60, TFRC, KPNB1, NSF, and SF3A3), which showed robust oncogenic features related to their genomic alterations, immunohistochemical changes, high interconnectivity with cancer driver genes (CDGs), and tumor vulnerabilities. Interestingly, some of these RBPs have never been studied in BC, but their oncogenic functions have been described in other cancer types. Subsequent analyses revealed PUF60 and SF3A3 as central elements of a spliceosome-related cluster involving RBPs and CDGs. Further research should focus on the mechanisms by which these proteins could promote breast tumorigenesis, with the potential to reveal new therapeutic pathways along with novel drug-development strategies.

Revisiting ZAR proteins: the understudied regulator of female fertility and beyond.

Putative RNA-binding proteins (RBPs), zygote arrested-1 (ZAR1), and ZAR2 (also known as ZAR1L), have been identified as maternal factors that mainly function in oogenesis and embryogenesis. Despite divergence in their spatio-temporal expression among species, the CxxC structure of the C-terminus of ZAR proteins is highly conserved and is reported to be the functional domain for the activity of the RBPs of ZAR proteins. In oocytes from Xenopus laevis and zebrafish, ZAR proteins have been reported to bind to maternal transcripts and inhibit translation in immature growing oocytes, whereas in fully grown mouse oocytes, they promote the translation during meiotic maturation. Thus, ZAR1 and ZAR2 may be required for the maternal-to-zygotic transition by stabilizing the maternal transcriptome in oocytes with partial functional redundancy. In addition, recent studies have suggested non-ovarian expression and function of ZAR proteins, particularly their involvement in tumorigenesis. ZAR proteins are potentially associated with tumor suppressors and can serve as epigenetically inactivated cancer biomarkers. In this review, studies on Zar1/2 are systematically summarized, and some issues that require discussion and further investigation are introduced as perspectives.

The mRNA-binding proteome of a critical phase transition during Arabidopsis seed germination.

Summary Arabidopsis thaliana seed germination is marked by extensive translational control at two critical phase transitions. The first transition refers to the start of hydration, the hydration translational shift. The second shift, the germination translational shift (GTS) is the phase between testa rupture and radicle protrusion at which the seed makes the all or nothing decision to germinate.The mechanism behind the translational regulation at these phase transitions is unknown. RNA binding proteins (RBPs) are versatile players in the post‐transcriptional control of messenger RNAs (mRNAs) and as such candidates for regulating translation during seed germination.Here, we report the mRNA binding protein repertoire of seeds during the GTS. Thirty seed specific RBPs and 22 dynamic RBPs were identified during the GTS, like the putative RBP Vacuolar ATPase subunit A and RBP HSP101. Several stress granule markers were identified in this study, which suggests that seeds are prepared to quickly adapt the translation of specific mRNAs in response to changes in environmental conditions during the GTS.Taken together this study provides a detailed insight into the world of RBPs during seed germination and their possible regulatory role during this developmentally regulated process.

Comparative genomics provides structural and functional insights into Bacteroides RNA biology.

Bacteria employ noncoding RNA molecules for a wide range of biological processes, including scaffolding large molecular complexes, catalyzing chemical reactions, defending against phages, and controlling gene expression. Secondary structures, binding partners, and molecular mechanisms have been determined for numerous small noncoding RNAs (sRNAs) in model aerobic bacteria. However, technical hurdles have largely prevented analogous analyses in the anaerobic gut microbiota. While experimental techniques are being developed to investigate the sRNAs of gut commensals, computational tools and comparative genomics can provide immediate functional insight. Here, using Bacteroides thetaiotaomicron as a representative microbiota member, we illustrate how comparative genomics improves our understanding of RNA biology in an understudied gut bacterium. We investigate putative RNA-binding proteins and predict a Bacteroides cold-shock protein homolog to have an RNA-related function. We apply an in silico protocol incorporating both sequence and structural analysis to determine the consensus structures and conservation of nine Bacteroides noncoding RNA families. Using structure probing, we validate and refine these predictions and deposit them in the Rfam database. Through synteny analyses, we illustrate how genomic coconservation can serve as a predictor of sRNA function. Altogether, this work showcases the power of RNA informatics for investigating the RNA biology of anaerobic microbiota members.

Lsm12 is an NAADP receptor and a two-pore channel regulatory protein required for calcium mobilization from acidic organelles

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+-mobilizing second messenger which uniquely mobilizes Ca2+ from acidic endolysosomal organelles. However, the molecular identity of the NAADP receptor remains unknown. Given the necessity of the endolysosomal two-pore channel (TPC1 or TPC2) in NAADP signaling, we performed affinity purification and quantitative proteomic analysis of the interacting proteins of NAADP and TPCs. We identified a Sm-like protein Lsm12 complexed with NAADP, TPC1, and TPC2. Lsm12 directly binds to NAADP via its Lsm domain, colocalizes with TPC2, and mediates the apparent association of NAADP to isolated TPC2 or TPC2-containing membranes. Lsm12 is essential and immediately participates in NAADP-evoked TPC activation and Ca2+ mobilization from acidic stores. These findings reveal a putative RNA-binding protein to function as an NAADP receptor and a TPC regulatory protein and provides a molecular basis for understanding the mechanisms of NAADP signaling.

Validation and classification of RNA binding proteins identified by mRNA interactome capture.

RNA binding proteins (RBPs) take part in all steps of the RNA life cycle and are often essential for cell viability. Most RBPs have a modular organization and comprise a set of canonical RNA binding domains. However, in recent years a number of high-throughput mRNA interactome studies on yeast, mammalian cell lines, and whole organisms have uncovered a multitude of novel mRNA interacting proteins that lack classical RNA binding domains. Whereas a few have been confirmed to be direct and functionally relevant RNA binders, biochemical and functional validation of RNA binding of most others is lacking. In this study, we used a combination of NMR spectroscopy and biochemical studies to test the RNA binding properties of six putative RBPs. Half of the analyzed proteins showed no interaction, whereas the other half displayed weak chemical shift perturbations upon titration with RNA. One of the candidates we found to interact weakly with RNA in vitro is Drosophila melanogaster end binding protein 1 (EB1), a master regulator of microtubule plus-end dynamics. Further analysis showed that EB1's RNA binding occurs on the same surface as that with which EB1 interacts with microtubules. RNA immunoprecipitation and colocalization experiments suggest that EB1 is a rather nonspecific, opportunistic RNA binder. Our data suggest that care should be taken when embarking on an RNA binding study involving these unconventional, novel RBPs, and we recommend initial and simple in vitro RNA binding experiments.

Focused CRISPR-Cas9 genetic screening reveals USO1 as a vulnerability in B-cell acute lymphoblastic leukemia

Post-transcriptional gene regulation, including that by RNA binding proteins (RBPs), has recently been described as an important mechanism in cancer. We had previously identified a set of RBPs that were highly dysregulated in B-cell acute lymphoblastic leukemia (B-ALL) with MLL translocations, which carry a poor prognosis. Here, we sought to functionally characterize these dysregulated RBP genes by performing a focused CRISPR dropout screen in B-ALL cell lines, finding dependencies on several genes including EIF3E, EPRS and USO1. Validating our findings, CRISPR/Cas9-mediated disruption of USO1 in MLL-translocated B-ALL cells reduced cell growth, promoted cell death, and altered the cell cycle. Transcriptomic analysis of USO1-deficient cells revealed alterations in pathways related to mTOR signaling, RNA metabolism, and targets of MYC. In addition, USO1-regulated genes from these experimental samples were significantly and concordantly correlated with USO1 expression in primary samples collected from B-ALL patients. Lastly, we found that loss of Uso1 inhibited colony formation of MLL-transformed in primary bone marrow cells from Cas9-EGFP mice. Together, our findings demonstrate an approach to performing focused sub-genomic CRISPR screens and highlight a putative RBP vulnerability in MLL-translocated B-ALL, thus identifying potential therapeutic targets in this disease.

The Putative RNA-Binding Protein Dri1 Promotes the Loading of Kinesin-14/Klp2 to the Mitotic Spindle and Is Sequestered into Heat-Induced Protein Aggregates in Fission Yeast.

Cells form a bipolar spindle during mitosis to ensure accurate chromosome segregation. Proper spindle architecture is established by a set of kinesin motors and microtubule-associated proteins. In most eukaryotes, kinesin-5 motors are essential for this process, and genetic or chemical inhibition of their activity leads to the emergence of monopolar spindles and cell death. However, these deficiencies can be rescued by simultaneous inactivation of kinesin-14 motors, as they counteract kinesin-5. We conducted detailed genetic analyses in fission yeast to understand the mechanisms driving spindle assembly in the absence of kinesin-5. Here, we show that deletion of the dri1 gene, which encodes a putative RNA-binding protein, can rescue temperature sensitivity caused by cut7-22, a fission yeast kinesin-5 mutant. Interestingly, kinesin-14/Klp2 levels on the spindles in the cut7 mutants were significantly reduced by the dri1 deletion, although the total levels of Klp2 and the stability of spindle microtubules remained unaffected. Moreover, RNA-binding motifs of Dri1 are essential for its cytoplasmic localization and function. We have also found that a portion of Dri1 is spatially and functionally sequestered by chaperone-based protein aggregates upon mild heat stress and limits cell division at high temperatures. We propose that Dri1 might be involved in post-transcriptional regulation through its RNA-binding ability to promote the loading of Klp2 on the spindle microtubules.

The Landscape of RNA-Protein Interactions in Plants: Approaches and Current Status

RNAs transmit information from DNA to encode proteins that perform all cellular processes and regulate gene expression in multiple ways. From the time of synthesis to degradation, RNA molecules are associated with proteins called RNA-binding proteins (RBPs). The RBPs play diverse roles in many aspects of gene expression including pre-mRNA processing and post-transcriptional and translational regulation. In the last decade, the application of modern techniques to identify RNA–protein interactions with individual proteins, RNAs, and the whole transcriptome has led to the discovery of a hidden landscape of these interactions in plants. Global approaches such as RNA interactome capture (RIC) to identify proteins that bind protein-coding transcripts have led to the identification of close to 2000 putative RBPs in plants. Interestingly, many of these were found to be metabolic enzymes with no known canonical RNA-binding domains. Here, we review the methods used to analyze RNA–protein interactions in plants thus far and highlight the understanding of plant RNA–protein interactions these techniques have provided us. We also review some recent protein-centric, RNA-centric, and global approaches developed with non-plant systems and discuss their potential application to plants. We also provide an overview of results from classical studies of RNA–protein interaction in plants and discuss the significance of the increasingly evident ubiquity of RNA–protein interactions for the study of gene regulation and RNA biology in plants.

The Experimental Proteome of Leishmania infantum Promastigote and Its Usefulness for Improving Gene Annotations.

Leishmania infantum causes visceral leishmaniasis (kala-azar), the most severe form of leishmaniasis, which is lethal if untreated. A few years ago, the re-sequencing and de novo assembling of the L. infantum (JPCM5 strain) genome was accomplished, and now we aimed to describe and characterize the experimental proteome of this species. In this work, we performed a proteomic analysis from axenic cultured promastigotes and carried out a detailed comparison with other Leishmania experimental proteomes published to date. We identified 2352 proteins based on a search of mass spectrometry data against a database built from the six-frame translated genome sequence of L. infantum. We detected many proteins belonging to organelles such as glycosomes, mitochondria, or flagellum, as well as many metabolic enzymes and many putative RNA binding proteins and molecular chaperones. Moreover, we listed some proteins presenting post-translational modifications, such as phosphorylations, acetylations, and methylations. On the other hand, the identification of peptides mapping to genomic regions previously annotated as non-coding allowed for the correction of annotations, leading to the N-terminal extension of protein sequences and the uncovering of eight novel protein-coding genes. The alliance of proteomics, genomics, and transcriptomics has resulted in a powerful combination for improving the annotation of the L. infantum reference genome.

Global cataloguing of variations in untranslated regions of viral genome and prediction of key host RNA binding protein-microRNA interactions modulating genome stability in SARS-CoV-2.

BackgroundThe world is going through the critical phase of COVID-19 pandemic, caused by human coronavirus, SARS-CoV-2. Worldwide concerted effort to identify viral genomic changes across different sub-types has identified several strong changes in the coding region. However, there have not been many studies focusing on the variations in the 5’ and 3’ untranslated regions and their consequences. Considering the possible importance of these regions in host mediated regulation of viral RNA genome, we wanted to explore the phenomenon.MethodsTo have an idea of the global changes in 5’ and 3’-UTR sequences, we downloaded 8595 complete and high-coverage SARS-CoV-2 genome sequence information from human host in FASTA format from Global Initiative on Sharing All Influenza Data (GISAID) from 15 different geographical regions. Next, we aligned them using Clustal Omega software and investigated the UTR variants. We also looked at the putative host RNA binding protein (RBP) and microRNA binding sites in these regions by ‘RBPmap’ and ‘RNA22 v2’ respectively. Expression status of selected RBPs and microRNAs were checked in lungs tissue.ResultsWe identified 28 unique variants in SARS-CoV-2 UTR region based on a minimum variant percentage cut-off of 0.5. Along with 241C>T change the important 5’-UTR change identified was 187A>G, while 29734G>C, 29742G>A/T and 29774C>T were the most familiar variants of 3’UTR among most of the continents. Furthermore, we found that despite the variations in the UTR regions, binding of host RBP to them remains mostly unaltered, which further influenced the functioning of specific miRNAs.ConclusionOur results, shows for the first time in SARS-Cov-2 infection, a possible cross-talk between host RBPs-miRNAs and viral UTR variants, which ultimately could explain the mechanism of escaping host RNA decay machinery by the virus. The knowledge might be helpful in developing anti-viral compounds in future.

Comparative analysis of RNA-binding proteomes under Arabidopsis thaliana-Pst DC3000-PAMP interaction by orthogonal organic phase separation

RNA-binding proteins (RBPs) are pivotal participants in post-transcriptional gene regulation. They interact with RNA directly to perform several post-transcriptional RNA regulatory functions or direct metabolic processes. Despite the essential importance, the understanding of plant RBPs is elementary, which derives mainly from other kingdoms via bioinformatic extrapolation or mRNA-binding proteins captured through UV crosslinked method. Recently, orthogonal organic phase separation (OOPS) method for RBP identification has been used in mammals and Escherichia coli. And plentiful RBPs were enriched without molecular tagging or capture of polyadenylated RNA in an unbiased way. In our study, OOPS was conducted on Arabidopsis and 468 RBPs were discovered including 244 putative RBPs. There were 17 peroxidases in 232 RBPs with enzymatic activities. In addition, Arabidopsis thaliana-Pst DC3000-chitinpentaose interaction system was chosen to explore whether OOPS can be used to dig specific RBPs under special physiological conditions. Eighty-four differential RBPs in this system were found and some of them involved in reactive oxygen species (ROS) metabolic pathway. These results showed OOPS can be applied to plants successfully and would be a useful method to identify RBPomes and specific RBPs.

BraInMap Elucidates the 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 their underlying molecular networks remains limited. Here, we applied multi-dimensional biochemical fractionation with mass spectrometry and machine learning to survey endogenous macromolecules across the adult mouse brain. We defined a global "interactome" comprising over one thousand multi-protein complexes. These include hundreds of brain-selective assemblies that have distinct physical and functional attributes, show regional and cell-type specificity, and have links to core neurological processes and disorders. Using reciprocal pull-downs and a transgenic model, we validated a putative 28-member RNA-binding protein complex associated with amyotrophic lateral sclerosis, suggesting a coordinated function in alternative splicing in disease progression. This brain interaction map (BraInMap) resource facilitates mechanistic exploration of the unique molecular machinery driving core cellular processes of the central nervous system. It is publicly available and can be explored here https://www.bu.edu/dbin/cnsb/mousebrain/.

TIMP1 intron 3 retention is a marker of colon cancer progression controlled by hnRNPA1.

We previously reported a 40-transcripts signature marking the normal mucosa to colorectal adenocarcinoma transition. Eight of these mRNAs also showed splicing alterations, including a specific intron 3 retention in tissue metalloprotease inhibitor I (TIMP1), which decreased during the early steps of colorectal cancer progression. To decipher the mechanism of intron 3 retention/splicing, we first searched for putative RNA binding protein binding sites onto the TIMP1 sequence. We identified potential serine arginine rich splicing factor 1 (SRSF1) and heterogeneous nuclear RiboNucleoProtein A1 (hnRNPA1) binding sites at the end of intron 3 and the beginning of exon 4, respectively. RNA immunoprecipitation showed that hnRNPA1, but not SRSF1 could bind to the corresponding region in TIMP1 pre-mRNA in live cells. Furthermore, using a TIMP1-based ex vivo minigene approach, together with a plasmon resonance in vitro RNA binding assay, we confirmed that hnRNPA1 could indeed bind to wild type TIMP1 exon 4 pre-mRNA and control TMP1 intron 3 splicing, the interaction being abolished in presence of a mutant sequence that disrupted this site. These results indicated that hnRNPA1, upon binding to TIMP1 exon 4, was a positive regulator of intron 3 splicing. We propose that this TIMP1-intron 3 + transcript belongs to the class of nuclear transcripts with "detained" introns, an abundant molecular class, including in cancer.