mRNA-bound Proteome Research Articles

The mRNA-bound Proteome of Leishmania mexicana: Novel Genetic Insight into an Ancient Parasite

Leishmania parasite infections, termed the leishmaniases, cause significant global infectious disease burden. The lifecycle of the parasite embodies three main stages that require precise coordination of gene regulation to survive environmental shifts between sandfly and mammalian hosts. Constitutive transcription in kinetoplastid parasites means that gene regulation is overwhelmingly reliant on post-transcriptional mechanisms, yet strikingly few Leishmania trans-regulators are known. Using optimized crosslinking and deep, quantified mass spectrometry, we present a comprehensive analysis of 1400 mRNA binding proteins (mRBPs) and whole cell proteomes from the three main Leishmania lifecycle stages. Supporting the validity, although the crosslinked RBPome is magnitudes more enriched, the protein identities of the crosslinked and non-crosslinked RBPomes were nearly identical. Moreover, multiple candidate RBPs were endogenously tagged and found to associate with discrete mRNA target pools in a stage-specific manner. Results indicate that in L. mexicana parasites, mRNA levels are not a strong predictor of the whole cell expression or RNA binding potential of encoded proteins. Evidence includes a low correlation between transcript and corresponding protein expression and stage-specific variation in protein expression versus RNA binding potential. Unsurprisingly, RNA binding protein enrichment correlates strongly with relative replication efficiency of the specific lifecycle stage. Our study is the first to quantitatively define and compare the mRBPome of multiple stages in kinetoplastid parasites. It provides novel, in-depth insight into the trans-regulatory mRNA:Protein (mRNP) complexes that drive Leishmania parasite lifecycle progression.

MRNA Interactome Capture from Plant Protoplasts.

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called "mRNA interactome capture," which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.

Dynamic RNA-protein interactions underlie the zebrafish maternal-to-zygotic transition.

During the maternal-to-zygotic transition (MZT), transcriptionally silent embryos rely on post-transcriptional regulation of maternal mRNAs until zygotic genome activation (ZGA). RNA-binding proteins (RBPs) are important regulators of post-transcriptional RNA processing events, yet their identities and functions during developmental transitions in vertebrates remain largely unexplored. Using mRNA interactome capture, we identified 227 RBPs in zebrafish embryos before and during ZGA, hereby named the zebrafish MZT mRNA-bound proteome. This protein constellation consists of many conserved RBPs, some of which are potential stage-specific mRNA interactors that likely reflect the dynamics of RNA–protein interactions during MZT. The enrichment of numerous splicing factors like hnRNP proteins before ZGA was surprising, because maternal mRNAs were found to be fully spliced. To address potentially unique roles of these RBPs in embryogenesis, we focused on Hnrnpa1. iCLIP and subsequent mRNA reporter assays revealed a function for Hnrnpa1 in the regulation of poly(A) tail length and translation of maternal mRNAs through sequence-specific association with 3′ UTRs before ZGA. Comparison of iCLIP data from two developmental stages revealed that Hnrnpa1 dissociates from maternal mRNAs at ZGA and instead regulates the nuclear processing of pri-mir-430 transcripts, which we validated experimentally. The shift from cytoplasmic to nuclear RNA targets was accompanied by a dramatic translocation of Hnrnpa1 and other pre-mRNA splicing factors to the nucleus in a transcription-dependent manner. Thus, our study identifies global changes in RNA–protein interactions during vertebrate MZT and shows that Hnrnpa1 RNA-binding activities are spatially and temporally coordinated to regulate RNA metabolism during early development.

UV crosslinked mRNA-binding proteins captured from leaf mesophyll protoplasts.

BackgroundThe complexity of RNA regulation is one of the current frontiers in animal and plant molecular biology research. RNA-binding proteins (RBPs) are characteristically involved in post-transcriptional gene regulation through interaction with RNA. Recently, the mRNA-bound proteome of mammalian cell lines has been successfully cataloged using a new method called interactome capture. This method relies on UV crosslinking of proteins to RNA, purifying the mRNA using complementary oligo-dT beads and identifying the crosslinked proteins using mass spectrometry. We describe here an optimized system of mRNA interactome capture for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type often used in functional cellular assays.ResultsWe established the conditions for optimal protein yield, namely the amount of starting tissue, the duration of UV irradiation and the effect of UV intensity. We demonstrated high efficiency mRNA-protein pull-down by oligo-d(T)25 bead capture. Proteins annotated to have RNA-binding capacity were overrepresented in the obtained medium scale mRNA-bound proteome, indicating the specificity of the method and providing in vivo UV crosslinking experimental evidence for several candidate RBPs from leaf mesophyll protoplasts.ConclusionsThe described method, applied to plant cells, allows identifying proteins as having the capacity to bind mRNA directly. The method can now be scaled and applied to other plant cell types and species to contribute to the comprehensive description of the RBP proteome of plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-016-0142-6) contains supplementary material, which is available to authorized users.

Gene expression regulatory networks in Trypanosoma brucei: insights into the role of the mRNA-binding proteome.

Control of gene expression at the post-transcriptional level is essential in all organisms, and RNA-binding proteins play critical roles from mRNA synthesis to decay. To fully understand this process, it is necessary to identify the complete set of RNA-binding proteins and the functional consequences of the protein-mRNA interactions. Here, we provide an overview of the proteins that bind to mRNAs and their functions in the pathogenic bloodstream form of Trypanosoma brucei. We describe the production of a small collection of open-reading frames encoding proteins potentially involved in mRNA metabolism. With this ORFeome collection, we used tethering to screen for proteins that play a role in post-transcriptional control. A yeast two-hybrid screen showed that several of the discovered repressors interact with components of the CAF1/NOT1 deadenylation complex. To identify the RNA-binding proteins, we obtained the mRNA-bound proteome. We identified 155 high-confidence candidates, including many not previously annotated as RNA-binding proteins. Twenty seven of these proteins affected reporter expression in the tethering screen. Our study provides novel insights into the potential trypanosome mRNPs composition, architecture and function.

The Cellular EJC Interactome Reveals Higher-Order mRNP Structure and an EJC-SR Protein Nexus

In addition to sculpting eukaryotic transcripts by removing introns, pre-mRNA splicing greatly impacts protein composition of the emerging mRNP. The exon junction complex (EJC), deposited upstream of exon-exon junctions after splicing, is a major constituent of spliced mRNPs. Here, we report comprehensive analysis of the endogenous human EJC protein and RNA interactomes. We confirm that the major "canonical" EJC occupancy site in vivo lies 24 nucleotides upstream of exon junctions and that the majority of exon junctions carry an EJC. Unexpectedly, we find that endogenous EJCs multimerize with one another and with numerous SR proteins to form megadalton sized complexes in which SR proteins are super-stoichiometric to EJC core factors. This tight physical association may explain known functional parallels between EJCs and SR proteins. Further, their protection of long mRNA stretches from nuclease digestion suggests that endogenous EJCs and SR proteins cooperate to promote mRNA packaging and compaction.

The mRNA-Bound Proteome and Its Global Occupancy Profile on Protein-Coding Transcripts

Protein-RNA interactions are fundamental to core biological processes, such as mRNA splicing, localization, degradation, and translation. We developed a photoreactive nucleotide-enhanced UV crosslinking and oligo(dT) purification approach to identify the mRNA-bound proteome using quantitative proteomics and to display the protein occupancy on mRNA transcripts by next-generation sequencing. Application to a human embryonic kidney cell line identified close to 800 proteins. To our knowledge, nearly one-third were not previously annotated as RNA binding, and about 15% were not predictable by computational methods to interact with RNA. Protein occupancy profiling provides a transcriptome-wide catalog of potential cis-regulatory regions on mammalian mRNAs and showed that large stretches in 3' UTRs can be contacted by the mRNA-bound proteome, with numerous putative binding sites in regions harboring disease-associated nucleotide polymorphisms. Our observations indicate the presence of a large number of mRNA binders with diverse molecular functions participating in combinatorial posttranscriptional gene-expression networks.