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.

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.

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.

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.

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.

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.

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.

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.

A novel role for Celf1 in vegetal RNA localization during Xenopus oogenesis.

The localization of certain mRNAs to the vegetal cortex of Xenopus oocytes is of crucial importance for germ cell development and early embryonic patterning. Vegetal RNA localization is mediated by cis-acting RNA localization elements (LE). Several proteins assemble on the RNA LE and direct transport to the vegetal cortex. Although a number of localization RNP components have been identified, their full composition is unknown. In an RNA affinity purification approach, using the dead end 1 (dnd1) RNA LE, we identified Xenopus Celf1 as a novel component of vegetal localization RNP complexes. Celf1 is part of an RNP complex together with known vegetal localization factors and shows specific interactions with LEs from several but not all vegetally localizing RNAs. Immunostaining experiments reveal co-localization of Celf1 with vegetally localizing RNA and with known localization factors. Inhibition of Celf1 protein binding by localization element mutagenesis as well as Celf1 overexpression interfere with vegetal RNA localization. These results argue for a role of Celf1 in vegetal RNA localization during Xenopus oogenesis.

Crystal structures and RNA-binding properties of the RNA recognition motifs of heterogeneous nuclear ribonucleoprotein L: insights into its roles in alternative splicing regulation.

Heterogeneous nuclear ribonucleoprotein L (hnRNP L) is an abundant RNA-binding protein implicated in many bioprocesses, including pre-mRNA processing, mRNA export of intronless genes, internal ribosomal entry site-mediated translation, and chromatin modification. It contains four RNA recognition motifs (RRMs) that bind with CA repeats or CA-rich elements. In this study, surface plasmon resonance spectroscopy assays revealed that all four RRM domains contribute to RNA binding. Furthermore, we elucidated the crystal structures of hnRNP L RRM1 and RRM34 at 2.0 and 1.8 Å, respectively. These RRMs all adopt the typical ß1α1ß2ß3α2ß4 topology, except for an unusual fifth ß-strand in RRM3. RRM3 and RRM4 interact intimately with each other mainly through helical surfaces, leading the two ß-sheets to face opposite directions. Structure-based mutations and surface plasmon resonance assay results suggested that the ß-sheets of RRM1 and RRM34 are accessible for RNA binding. FRET-based gel shift assays (FRET-EMSA) and steady-state FRET assays, together with cross-linking and dynamic light scattering assays, demonstrated that hnRNP L RRM34 facilitates RNA looping when binding to two appropriately separated binding sites within the same target pre-mRNA. EMSA and isothermal titration calorimetry binding studies with in vivo target RNA suggested that hnRNP L-mediated RNA looping may occur in vivo. Our study provides a mechanistic explanation for the dual functions of hnRNP L in alternative splicing regulation as an activator or repressor.

Structural analyses of the CRISPR protein Csc2 reveal the RNA-binding interface of the type I-D Cas7 family.

Upon pathogen invasion, bacteria and archaea activate an RNA-interference-like mechanism termed CRISPR (clustered regularly interspaced short palindromic repeats). A large family of Cas (CRISPR-associated) proteins mediates the different stages of this sophisticated immune response. Bioinformatic studies have classified the Cas proteins into families, according to their sequences and respective functions. These range from the insertion of the foreign genetic elements into the host genome to the activation of the interference machinery as well as target degradation upon attack. Cas7 family proteins are central to the type I and type III interference machineries as they constitute the backbone of the large interference complexes. Here we report the crystal structure of Thermofilum pendens Csc2, a Cas7 family protein of type I-D. We found that Csc2 forms a core RRM-like domain, flanked by three peripheral insertion domains: a lid domain, a Zinc-binding domain and a helical domain. Comparison with other Cas7 family proteins reveals a set of similar structural features both in the core and in the peripheral domains, despite the absence of significant sequence similarity. T. pendens Csc2 binds single-stranded RNA in vitro in a sequence-independent manner. Using a crosslinking - mass-spectrometry approach, we mapped the RNA-binding surface to a positively charged surface patch on T. pendens Csc2. Thus our analysis of the key structural and functional features of T. pendens Csc2 highlights recurring themes and evolutionary relationships in type I and type III Cas proteins.

Structure and RNA-binding properties of the type III-A CRISPR-associated protein Csm3.

The prokaryotic adaptive immune system is based on the incorporation of genome fragments of invading viral genetic elements into clusters of regulatory interspaced short palindromic repeats (CRISPRs). The CRISPR loci are transcribed and processed into crRNAs, which are then used to target the invading nucleic acid for degradation. The large family of CRISPR-associated (Cas) proteins mediates this interference response. We have characterized Methanopyrus kandleri Csm3, a protein of the type III-A CRISPR-Cas complex. The 2.4 Å resolution crystal structure shows an elaborate four-domain fold organized around a core RRM-like domain. The overall architecture highlights the structural homology to Cas7, the Cas protein that forms the backbone of type I interference complexes. Csm3 binds unstructured RNAs in a sequence non-specific manner, suggesting that it interacts with the variable spacer sequence of the crRNA. The structural and biochemical data provide insights into the similarities and differences in this group of Cas proteins.

CRISPR-Cas: evolution of an RNA-based adaptive immunity system in prokaryotes.

The CRISPR-Cas (clustered regularly interspaced short palindromic repeats, CRISPR-associated genes) is an adaptive immunity system in bacteria and archaea that functions via a distinct self-non-self recognition mechanism that is partially analogous to the mechanism of eukaryotic RNA interference (RNAi). The CRISPR-Cas system incorporates fragments of virus or plasmid DNA into the CRISPR repeat cassettes and employs the processed transcripts of these spacers as guide RNAs to cleave the cognate foreign DNA or RNA. The Cas proteins, however, are not homologous to the proteins involved in RNAi and comprise numerous, highly diverged families. The majority of the Cas proteins contain diverse variants of the RNA recognition motif (RRM), a widespread RNA-binding domain. Despite the fast evolution that is typical of the cas genes, the presence of diverse versions of the RRM in most Cas proteins provides for a simple scenario for the evolution of the three distinct types of CRISPR-cas systems. In addition to several proteins that are directly implicated in the immune response, the cas genes encode a variety of proteins that are homologous to prokaryotic toxins that typically possess nuclease activity. The predicted toxins associated with CRISPR-Cas systems include the essential Cas2 protein, proteins of COG1517 that, in addition to a ligand-binding domain and a helix-turn-helix domain, typically contain different nuclease domains and several other predicted nucleases. The tight association of the CRISPR-Cas immunity systems with predicted toxins that, upon activation, would induce dormancy or cell death suggests that adaptive immunity and dormancy/suicide response are functionally coupled. Such coupling could manifest in the persistence state being induced and potentially providing conditions for more effective action of the immune system or in cell death being triggered when immunity fails.

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.

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.

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.

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

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

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.