In Vivo and In Vitro Characterization of the RNA Binding Capacity of SETD1A (KMT2F).

For several histone lysine methyltransferases (HKMTs), RNA binding has been already shown to be a functionally relevant feature, but detailed information on the RNA interactome of these proteins is not always known. Of the six human KMT2 proteins responsible for the methylation of the H3K4 residue, two-SETD1A and SETD1B-contain RNA recognition domains (RRMs). Here we investigated the RNA binding capacity of SETD1A and identified a broad range of interacting RNAs within HEK293T cells. Our analysis revealed that similar to yeast Set1, SETD1A is also capable of binding several coding and non-coding RNAs, including RNA species related to RNA processing. We also show direct RNA binding activity of the individual RRM domain in vitro, which is in contrast with the RRM domain found in yeast Set1. Structural modeling revealed important details on the possible RNA recognition mode of SETD1A and highlighted some fundamental differences between SETD1A and Set1, explaining the differences in the RNA binding capacity of their respective RRMs.

Insight into the Structural Basis for Dual Nucleic Acid-Recognition by the Scaffold Attachment Factor B2 Protein.

The family of scaffold attachment factor B (SAFB) proteins comprises three members and was first identified as binders of the nuclear matrix/scaffold. Over the past two decades, SAFBs were shown to act in DNA repair, mRNA/(l)ncRNA processing and as part of protein complexes with chromatin-modifying enzymes. SAFB proteins are approximately 100 kDa-sized dual nucleic acid-binding proteins with dedicated domains in an otherwise largely unstructured context, but whether and how they discriminate DNA and RNA binding has remained enigmatic. We here provide the SAFB2 DNA- and RNA-binding SAP and RRM domains in their functional boundaries and use solution NMR spectroscopy to ascribe DNA- and RNA-binding functions. We give insight into their target nucleic acid preferences and map the interfaces with respective nucleic acids on sparse data-derived SAP and RRM domain structures. Further, we provide evidence that the SAP domain exhibits intra-domain dynamics and a potential tendency to dimerize, which may expand its specifically targeted DNA sequence range. Our data provide a first molecular basis of and a starting point towards deciphering DNA- and RNA-binding functions of SAFB2 on the molecular level and serve a basis for understanding its localization to specific regions of chromatin and its involvement in the processing of specific RNA species.

1H, 13C and 15N resonance assignments of stress granule key component G3BP1 RRM domain.

G3BP1 exists as a helicase and one of the core components in stress granules, which are associated with a variety of neurodegenerative diseases. Its RNA recognition motif (RRM) domain performs the paramount function of binding mRNA. Here we report the resonance assignment of human G3BP1 RRM domain to understand its structure-function relationship.

Structural and dynamic studies of the human RNA binding protein RBM3 reveals the molecular basis of its oligomerization and RNA recognition.

Human RNA-binding motif 3 protein (RBM3) is a cold-shock protein which functions in various aspects of global protein synthesis, cell proliferation and apoptosis by interacting with the components of basal translational machinery. RBM3 plays important roles in tumour progression and cancer metastasis, and also has been shown to be involved in neuroprotection and endoplasmic reticulum stress response. Here, we have solved the solution NMR structure of the N-terminal 84 residue RNA recognition motif (RRM) of RBM3. The remaining residues are rich in RGG and YGG motifs and are disordered. The RRM domain adopts a ßαßßαß topology, which is found in many RNA-binding proteins. NMR-monitored titration experiments and molecular dynamic simulations show that the beta-sheet and two loops form the RNA-binding interface. Hydrogen bond, pi-pi and pi-cation are the key interactions between the RNA and the RRM domain. NMR, size exclusion chromatography and chemical cross-linking experiments show that RBM3 forms oligomers in solution, which is favoured by decrease in temperature, thus, potentially linking it to its function as a cold-shock protein. Temperature-dependent NMR studies revealed that oligomerization of the RRM domain occurs via nonspecific interactions. Overall, this study provides the detailed structural analysis of RRM domain of RBM3, its interaction with RNA and the molecular basis of its temperature-dependent oligomerization.

Structural basis of PETISCO complex assembly during piRNA biogenesis in C. elegans.

Piwi-interacting RNAs (piRNAs) constitute a class of small RNAs that bind PIWI proteins and are essential to repress transposable elements in the animal germline, thereby promoting genome stability and maintaining fertility. C. elegans piRNAs (21U RNAs) are transcribed individually from minigenes as precursors that require 5' and 3' processing. This process depends on the PETISCO complex, consisting of four proteins: IFE-3, TOFU-6, PID-3, and ERH-2. We used biochemical and structural biology approaches to characterize the PETISCO architecture and its interaction with RNA, together with its effector proteins TOST-1 and PID-1. These two proteins define different PETISCO functions: PID-1 governs 21U processing, whereas TOST-1 links PETISCO to an unknown process essential for early embryogenesis. Here, we show that PETISCO forms an octameric assembly with each subunit present in two copies. Determination of structures of the TOFU-6/PID-3 and PID-3/ERH-2 subcomplexes, supported by in vivo studies of subunit interaction mutants, allows us to propose a model for the formation of the TOFU-6/PID-3/ERH-2 core complex and its functionality in germ cells and early embryos. Using NMR spectroscopy, we demonstrate that TOST-1 and PID-1 bind to a common surface on ERH-2, located opposite its PID-3 binding site, explaining how PETISCO can mediate different cellular roles.

Structures and target RNA preferences of the RNA-binding protein family of IGF2BPs: An overview.

Insulin-like growth factor 2 mRNA-binding proteins (IMPs, IGF2BPs) act in mRNA transport and translational control but are oncofetal tumor marker proteins. The IMP protein family represents a number of bona fide multi-domain RNA-binding proteins with up to six RNA-binding domains, resulting in a high complexity of possible modes of interactions with target mRNAs. Their exact mechanism in stability control of oncogenic mRNAs is only partially understood. Our and other laboratories' recent work has significantly pushed the understanding of IMP protein specificities both toward RNA engagement and between each other from NMR and crystal structures serving the basis for systematic biochemical and functional investigations. We here summarize the known structural and biochemical information about IMP RNA-binding domains and their RNA preferences. The article also touches on the respective roles of RNA secondary and protein tertiary structures for specific RNA-protein complexes, including the limited knowledge about IMPs' protein-protein interactions, which are often RNA mediated.

Resonance assignments of La protein RRM domain from Trypanosoma brucei.

The autoantigen La protein is a conserved component of eukaryotic ribonucleoprotein complexes that binds the 3' poly(U) sequences of nascent RNA polymerase III transcripts to assist folding and maturation. This specific recognition is mediated by the N-terminal domain (NTD) of La, which comprises a La motif and an RNA recognition motif (RRM). Here, we report near complete 1H, 13C and 15N backbone and sidechain assignments for the RRM domain of La protein from Trypanosoma brucei.

Corrigendum: Structural Insights Into TDP-43 and Effects of Post-translational Modifications.

[This corrects the article DOI: 10.3389/fnmol.2019.00301.].

Modulation of histone modifications and G-quadruplex structures by G-quadruplex-binding proteins.

The functions of local conformations of non-B form DNA and RNA, such as the G-quadruplex, are thought to be regulated by their specific binding proteins. They regulate the formation of G-quadruplexes in cells and affect the biological functions of G-quadruplexes. Recent studies reported that G-quadruplexes regulate epigenetics through these G-quadruplex binding proteins. We discuss regulation of histone modifications through G-quadruplex RNA and its binding proteins which modulate the G-quadruplex conformations. G-quadruplex RNA is involved in telomere maintenance and transcription via histone modification. Furthermore, G-quadruplex binding proteins regulate formation and biological functions of G-quadruplexes through regulating their folding or unfolding. In this review, we will focus on the G-quadruplex binding proteins containing RRM and RGG domains.

NMR Fragment-Based Screening against Tandem RNA Recognition Motifs of TDP-43.

The TDP-43 is originally a nuclear protein but translocates to the cytoplasm in the pathological condition. TDP-43, as an RNA-binding protein, consists of two RNA Recognition Motifs (RRM1 and RRM2). RRMs are known to involve both protein-nucleotide and protein-protein interactions and mediate the formation of stress granules. Thus, they assist the entire TDP-43 protein with participating in neurodegenerative and cancer diseases. Consequently, they are potential therapeutic targets. Protein-observed and ligand-observed nuclear magnetic resonance (NMR) spectroscopy were used to uncover the small molecule inhibitors against the tandem RRM of TDP-43. We identified three hits weakly binding the tandem RRMs using the ligand-observed NMR fragment-based screening. The binding topology of these hits is then depicted by chemical shift perturbations (CSP) of the 15N-labeled tandem RRM and RRM2, respectively, and modeled by the CSP-guided High Ambiguity Driven biomolecular DOCKing (HADDOCK). These hits mainly bind to the RRM2 domain, which suggests the druggability of the RRM2 domain of TDP-43. These hits also facilitate further studies regarding the hit-to-lead evolution against the TDP-43 RRM domain.

A telomerase subunit homolog La protein from Trypanosoma brucei plays an essential role in ribosomal biogenesis.

The autoantigen La protein is an important component of telomerase and a predominantly nuclear phosphoprotein. As a telomerase subunit, La protein associates with the telomerase ribonucleoprotein and influences telomere length. In the reverse transcription, La protein stimulates enzymatic activity and increases repeated addition processivity of telomerase. As nuclear phosphoprotein, La protein binds the 3' poly(U)-rich elements of nascent RNA polymerase III transcripts to facilitate its correct folding and maturation. In this work, we identified a La protein homolog (TbLa) from Trypanosoma brucei (T. brucei). We revealed that TbLa interacts with ribosome-associated protein P34/P37, 40S ribosomal protein SA, and 60S ribosomal subunit L5 in T. brucei. In the interactions between TbLa protein and (P34/P37)/L5/SA, RNA recognition motif (RRM) domain of TbLa was indicated to make the major contribution to the processes. We determined the solution structure of TbLa RRM domain. NMR chemical shift perturbations revealed that the positively charged RNA-binding pocket of TbLa RRM domain is responsible for its interaction with ribosomal and ribosome-associated proteins P37/L5/SA. Furthermore, depletion of TbLa affected the maturation process of 5S rRNA and ribosomal assembly, suggesting TbLa protein might play a significant role in the ribosomal biogenesis pathway in T. brucei. Taken together, our results provide a novel insight and structural basis for better understanding the roles of TbLa and RRM domain in ribosomal biogenesis in T. brucei. DATABASE: Structural data are available in the PDB under the accession number 5ZUH.

RRM Transcription Factors Interact with NLRs and Regulate Broad-Spectrum Blast Resistance in Rice.

Nucleotide-binding site leucine-rich repeat (NLR) receptors perceive pathogen effectors and trigger plant immunity. However, the mechanisms underlying NLR-triggered defense responses remain obscure. The recently discovered Pigm locus in rice encodes a cluster of NLRs, including PigmR, which confers broad-spectrum resistance to blast fungus. Here, we identify PIBP1 (PigmR-INTERACTING and BLAST RESISTANCE PROTEIN 1), an RRM (RNA-recognition motif) protein that specifically interacts with PigmR and other similar NLRs to trigger blast resistance. PigmR-promoted nuclear accumulation of PIBP1 ensures full blast resistance. We find that PIBP1 and a homolog, Os06 g02240, bind DNA and function as unconventional transcription factors at the promoters of the defense genes OsWAK14 and OsPAL1, activating their expression. Knockout of PIBP1 and Os06 g02240 greatly attenuated blast resistance. Collectively, our study discovers previously unappreciated RRM transcription factors that directly interact with NLRs to activate plant defense, establishing a direct link between transcriptional activation of immune responses with NLR-mediated pathogen perception.

Histone 2B monoubiquitination complex integrates transcript elongation with RNA processing at circadian clock and flowering regulators.

HISTONE MONOUBIQUITINATION1 (HUB1) and its paralog HUB2 act in a conserved heterotetrameric complex in the chromatin-mediated transcriptional modulation of developmental programs, such as flowering time, dormancy, and the circadian clock. The KHD1 and SPEN3 proteins were identified as interactors of the HUB1 and HUB2 proteins with in vitro RNA-binding activity. Mutants in SPEN3 and KHD1 had reduced rosette and leaf areas. Strikingly, in spen3 mutants, the flowering time was slightly, but significantly, delayed, as opposed to the early flowering time in the hub1-4 mutant. The mutant phenotypes in biomass and flowering time suggested a deregulation of their respective regulatory genes CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) and FLOWERING LOCUS C (FLC) that are known targets of the HUB1-mediated histone H2B monoubiquitination (H2Bub). Indeed, in the spen3-1 and hub1-4 mutants, the circadian clock period was shortened as observed by luciferase reporter assays, the levels of the CCA1α and CCA1ß splice forms were altered, and the CCA1 expression and H2Bub levels were reduced. In the spen3-1 mutant, the delay in flowering time was correlated with an enhanced FLC expression, possibly due to an increased distal versus proximal ratio of its antisense COOLAIR transcript. Together with transcriptomic and double-mutant analyses, our data revealed that the HUB1 interaction with SPEN3 links H2Bub during transcript elongation with pre-mRNA processing at CCA1 Furthermore, the presence of an intact HUB1 at the FLC is required for SPEN3 function in the formation of the FLC-derived antisense COOLAIR transcripts.

Structural Insights Into TDP-43 and Effects of Post-translational Modifications.

Transactive response DNA binding protein (TDP-43) is a key player in neurodegenerative diseases. In this review, we have gathered and presented structural information on the different regions of TDP-43 with high resolution structures available. A thorough understanding of TDP-43 structure, effect of modifications, aggregation and sites of localization is necessary as we develop therapeutic strategies targeting TDP-43 for neurodegenerative diseases. We discuss how different domains as well as post-translational modification may influence TDP-43 overall structure, aggregation and droplet formation. The primary aim of the review is to utilize structural insights as we develop an understanding of the deleterious behavior of TDP-43 and highlight locations of established and proposed post-translation modifications. TDP-43 structure and effect on localization is paralleled by many RNA-binding proteins and this review serves as an example of how structure may be modulated by numerous compounding elements.

Crystal structure of human Acinus RNA recognition motif domain.

Acinus is an abundant nuclear protein involved in apoptosis and splicing. It has been implicated in inducing apoptotic chromatin condensation and DNA fragmentation during programmed cell death. Acinus undergoes activation by proteolytic cleavage that produces a truncated p17 form that comprises only the RNA recognition motif (RRM) domain. We have determined the crystal structure of the human Acinus RRM domain (AcRRM) at 1.65 Å resolution. It shows a classical four-stranded antiparallel ß-sheet fold with two flanking α-helices and an additional, non-classical α-helix at the C-terminus, which harbors the caspase-3 target sequence that is cleaved during Acinus activation. In the structure, the C-terminal α-helix partially occludes the potential ligand binding surface of the ß-sheet and hypothetically shields it from non-sequence specific interactions with RNA. Based on the comparison with other RRM-RNA complex structures, it is likely that the C-terminal α-helix changes its conformation with respect to the RRM core in order to enable RNA binding by Acinus.

Leishmania braziliensis SCD6 and RBP42 proteins, two factors with RNA binding capacity.

BACKGROUND: The study of RNA binding proteins (RBPs) is of great relevance for understanding processes like post-transcriptional control of gene expression. The post-transcriptional mechanisms are particularly important in Leishmania parasites and related trypanosomatids since transcriptional regulation is almost absent in them. Thus, RBPs should be essential during the development of these parasites and for survival strategies against the adverse conditions that they face during their life-cycle. This work was aimed to do a structural and biochemical characterization of two Leishmania braziliensis proteins, which were previously found in pull-down assays using an HSP70 RNA as bait. At that time, these proteins were annotated as hypothetical proteins (LbrM.25.2210 and LbrM.30.3080) in the GeneDB database. RESULTS: Structural analysis indicated that these two proteins belong to evolutionarily conserved families; thus, they have been renamed accordingly as LbSCD6 (LbrM.25.2210) and LbRBP42 (LbrM.30.3080). We have demonstrated experimentally that these proteins are RBPs, in agreement with their structural features. Both proteins were able to bind to the complete 3' UTR-II region of HSP70-type II mRNA, and to an A + U rich element (ARE) present in that UTR. Cellular localization assays suggested that both proteins are mainly distributed in the cytoplasm of promastigotes growing at 26 °C, but they accumulate in foci around the nucleus when the parasites are under heat-shock conditions. Also, our study showed that steady-state levels of LbSCD6 and LbRBP42 transcripts decreased significantly during incubation of L. braziliensis promastigotes at heat-shock temperatures. However, in these conditions, the cellular content of both proteins remained unaltered. CONCLUSIONS: Our data suggest that LbSCD6 and LbRBP42, as occurs for their orthologues in other organisms, are involved in mRNA regulation, and probably they have a relevant role facing the stress conditions that L. braziliensis encounters during insect-to-mammalian transmission.

An RNA Recognition Motif-Containing Protein Functions in Meiotic Silencing by Unpaired DNA.

Meiotic silencing by unpaired DNA (MSUD) is a biological process that searches pairs of homologous chromosomes (homologs) for segments of DNA that are unpaired. Genes found within unpaired segments are silenced for the duration of meiosis. In this report, we describe the identification and characterization of Neurospora crassa sad-7, a gene that encodes a protein with an RNA recognition motif (RRM). Orthologs of sad-7 are found in a wide range of ascomycete fungi. In N. crassa, sad-7 is required for a fully efficient MSUD response to unpaired genes. Additionally, at least one parent must have a functional sad-7 allele for a cross to produce ascospores. Although sad-7-null crosses are barren, sad-7Δ strains grow at a wild-type (wt) rate and appear normal under vegetative growth conditions. With respect to expression, sad-7 is transcribed at baseline levels in early vegetative cultures, at slightly higher levels in mating-competent cultures, and is at its highest level during mating. These findings suggest that SAD-7 is specific to mating-competent and sexual cultures. Although the role of SAD-7 in MSUD remains elusive, green fluorescent protein (GFP)-based tagging studies place SAD-7 within nuclei, perinuclear regions, and cytoplasmic foci of meiotic cells. This localization pattern is unique among known MSUD proteins and raises the possibility that SAD-7 coordinates nuclear, perinuclear, and cytoplasmic aspects of MSUD.

RRM domain of Arabidopsis splicing factor SF1 is important for pre-mRNA splicing of a specific set of genes.

KEY MESSAGE: The RNA recognition motif of Arabidopsis splicing factor SF1 affects the alternative splicing of FLOWERING LOCUS M pre-mRNA and a heat shock transcription factor HsfA2 pre-mRNA. Splicing factor 1 (SF1) plays a crucial role in 3' splice site recognition by binding directly to the intron branch point. Although plant SF1 proteins possess an RNA recognition motif (RRM) domain that is absent in its fungal and metazoan counterparts, the role of the RRM domain in SF1 function has not been characterized. Here, we show that the RRM domain differentially affects the full function of the Arabidopsis thaliana AtSF1 protein under different experimental conditions. For example, the deletion of RRM domain influences AtSF1-mediated control of flowering time, but not the abscisic acid sensitivity response during seed germination. The alternative splicing of FLOWERING LOCUS M (FLM) pre-mRNA is involved in flowering time control. We found that the RRM domain of AtSF1 protein alters the production of alternatively spliced FLM-ß transcripts. We also found that the RRM domain affects the alternative splicing of a heat shock transcription factor HsfA2 pre-mRNA, thereby mediating the heat stress response. Taken together, our results suggest the importance of RRM domain for AtSF1-mediated alternative splicing of a subset of genes involved in the regulation of flowering and adaptation to heat stress.

Predicted RNA Binding Proteins Pes4 and Mip6 Regulate mRNA Levels, Translation, and Localization during Sporulation in Budding Yeast.

In response to starvation, diploid cells of Saccharomyces cerevisiae undergo meiosis and form haploid spores, a process collectively referred to as sporulation. The differentiation into spores requires extensive changes in gene expression. The transcriptional activator Ndt80 is a central regulator of this process, which controls many genes essential for sporulation. Ndt80 induces ∼300 genes coordinately during meiotic prophase, but different mRNAs within the NDT80 regulon are translated at different times during sporulation. The protein kinase Ime2 and RNA binding protein Rim4 are general regulators of meiotic translational delay, but how differential timing of individual transcripts is achieved was not known. This report describes the characterization of two related NDT80-induced genes, PES4 and MIP6, encoding predicted RNA binding proteins. These genes are necessary to regulate the steady-state expression, translational timing, and localization of a set of mRNAs that are transcribed by NDT80 but not translated until the end of meiosis II. Mutations in the predicted RNA binding domains within PES4 alter the stability of target mRNAs. PES4 and MIP6 affect only a small portion of the NDT80 regulon, indicating that they act as modulators of the general Ime2/Rim4 pathway for specific transcripts.

The RNA recognition motif protein CP33A is a global ligand of chloroplast mRNAs and is essential for plastid biogenesis and plant development.

Chloroplast RNA metabolism depends on a multitude of nuclear-encoded RNA-binding proteins (RBPs). Most known chloroplast RBPs address specific RNA targets and RNA-processing functions. However, members of the small chloroplast ribonucleoprotein family (cpRNPs) play a global role in processing and stabilizing chloroplast RNAs. Here, we show that the cpRNP CP33A localizes to a distinct sub-chloroplastic domain and is essential for chloroplast development. The loss of CP33A yields albino seedlings that exhibit aberrant leaf development and can only survive in the presence of an external carbon source. Genome-wide RNA association studies demonstrate that CP33A associates with all chloroplast mRNAs. For a given transcript, quantification of CP33A-bound versus free RNAs demonstrates that CP33A associates with the majority of most mRNAs analyzed. Our results further show that CP33A is required for the accumulation of a number of tested mRNAs, and is particularly relevant for unspliced and unprocessed precursor mRNAs. Finally, CP33A fails to associate with polysomes or to strongly co-precipitate with ribosomal RNA, suggesting that it defines a ribodomain that is separate from the chloroplast translation machinery. Collectively, these findings suggest that CP33A contributes to globally essential RNA processes in the chloroplasts of higher plants.