In plants, nucleotide-binding site and leucine-rich repeat proteins (NLRs) play pivotal roles in effector-triggered immunity (ETI). However, the precise mechanisms underlying NLR-mediated disease resistance remain elusive. Previous studies have demonstrated that the NLR gene pair Pik-H4 confers resistance to rice blast disease by interacting with the transcription factor OsBIHD1, consequently leading to the upregulation of hormone pathways. In the present study, we identified an RNA recognition motif (RRM) protein, OsRRM2, which interacted with Pik1-H4 and Pik2-H4 in vesicles and chloroplasts. OsRRM2 exhibited a modest influence on Pik-H4-mediated rice blast resistance by upregulating resistance genes and genes associated with chloroplast immunity. Moreover, the RNA-binding sequence of OsRRM2 was elucidated using systematic evolution of ligands by exponential enrichment. Transcriptome analysis further indicated that OsRRM2 promoted RNA editing of the chloroplastic gene ndhB. Collectively, our findings uncovered a chloroplastic RRM protein that facilitated the translocation of the NLR gene pair and modulated chloroplast immunity, thereby bridging the gap between ETI and chloroplast immunity.

SRSF1 governs splicing of over 1500 mRNA transcripts. SRSF1 contains two RNA-recognition motifs (RRMs) and a C-terminal Arg/Ser-rich region (RS). It has been thought that SRSF1 RRMs exclusively recognize single-stranded exonic splicing enhancers, while RS lacks RNA-binding specificity. With our success in solving the insolubility problem of SRSF1, we can explore the unknown RNA-binding landscape of SRSF1. We find that SRSF1 RS prefers purine over pyrimidine. Moreover, SRSF1 binds to the G-quadruplex (GQ) from the ARPC2 mRNA, with both RRMs and RS being crucial. Our binding assays show that the traditional RNA-binding sites on the RRM tandem and the Arg in RS are responsible for GQ binding. Interestingly, our FRET and circular dichroism data reveal that SRSF1 unfolds the ARPC2 GQ, with RS leading unfolding and RRMs aiding. Our saturation transfer difference NMR results discover that Arg residues in SRSF1 RS interact with the guanine base but not other nucleobases, underscoring the uniqueness of the Arg/guanine interaction. Our luciferase assays confirm that SRSF1 can alleviate the inhibitory effect of GQ on gene expression in the cell. Given the prevalence of RNA GQ and SR proteins, our findings unveil unexplored SR protein functions with broad implications in RNA splicing and translation.

The RNA recognition motif (RRM) is the most common RNA-binding protein domain identified in nature. However, RRM-containing proteins are only prevalent in eukaryotic phyla, in which they play central regulatory roles. Here, we engineered an orthogonal post-transcriptional control system of gene expression in the bacterium Escherichia coli with the mammalian RNA-binding protein Musashi-1, which is a stem cell marker with neurodevelopmental role that contains two canonical RRMs. In the circuit, Musashi-1 is regulated transcriptionally and works as an allosteric translation repressor thanks to a specific interaction with the N-terminal coding region of a messenger RNA and its structural plasticity to respond to fatty acids. We fully characterized the genetic system at the population and single-cell levels showing a significant fold change in reporter expression, and the underlying molecular mechanism by assessing the in vitro binding kinetics and in vivo functionality of a series of RNA mutants. The dynamic response of the system was well recapitulated by a bottom-up mathematical model. Moreover, we applied the post-transcriptional mechanism engineered with Musashi-1 to specifically regulate a gene within an operon, implement combinatorial regulation, and reduce protein expression noise. This work illustrates how RRM-based regulation can be adapted to simple organisms, thereby adding a new regulatory layer in prokaryotes for translation control.

β-Hairpin peptides with RNA-binding sequences mimicking the central two β-strands of the RNA recognition motif (RRM) protein domain have been observed to bind in a 2:1 fashion to a series of RNA homooligonucleotides in aqueous solution (PBS buffer, pH 7.40) with binding energies (-27 to -35 kJ mol-1) similar to those of full-size protein RRMs. The peptides display mild selectivities with respect to the binding of the different homooligomers. Binding studies in 500 mM magnesium chloride suggest that the complex formation is not predominantly driven by Coulombic attraction. These peptides represent a starting point for further studies of non-Coulombic binding of RNA by peptides and proteins, which is important in the context of contemporary biology, potential therapeutic applications, and prebiotic peptide-RNA interactions.

T-cell intracellular antigen-1 (TIA-1) is a key RNA-binding protein that participates in translation regulation and RNA splicing. TIA-1 undergoes liquid-liquid phase separation as a fundamental mechanism that enables the condensation of RNA and proteins into membraneless organelles called stress granules (SGs). However, this dynamic behavior can lead to aberrant fibril formation, implicated in neurodegenerative disorders, and must be tightly regulated. In this study, we investigated the role in the cell of histidine residues His94 and His96, responsible for Zn2+ binding. Using fluorescence microscopy, we found that the specific binding site formed by these residues is critical for SG assembly. Furthermore, it also plays a role maintaining the dynamic behavior of SG-assembled TIA-1. Collectively, our findings confirm the physiological relevance of TIA-1 His94 and His96 in the Zn2+ -mediated regulatory mechanism for protection against fibril formation in SGs.

XIST noncoding RNA promotes the initiation of X chromosome silencing by recruiting the protein SPEN to one X chromosome in female mammals. The SPEN protein is also called SHARP (SMRT and HDAC-associated repressor protein) and MINT (Msx-2 interacting nuclear target) in humans. SPEN recruits N-CoR2 and HDAC3 to initiate histone deacetylation on the X chromosome, leading to the formation of repressive chromatin marks and silencing gene expression. We dissected the contributions of different RNA and protein regions to the formation of a human XIST-SPEN complex in vitro and identified novel sequence and structure determinants that may contribute to X chromosome silencing initiation. Binding of SPEN to XIST RNA requires RRM 4 of the protein, in contrast to the requirement of RRM 3 and RRM 4 for specific binding to SRA RNA. Measurements of SPEN binding to full-length, dimeric, trimeric, or other truncated versions of the A-repeat region revealed that high-affinity binding of XIST to SPEN in vitro requires a minimum of four A-repeat segments. SPEN binding to XIST A-repeat RNA changes the accessibility of the RNA at specific nucleotide sequences, as indicated by changes in RNA reactivity through chemical structure probing. Based on computational modeling, we found that inter-repeat duplexes formed by multiple A-repeats can present an unpaired adenosine in the context of a double-stranded region of RNA. The presence of this specific combination of sequence and structural motifs correlates with high-affinity SPEN binding in vitro. These data provide new information on the molecular basis of the XIST and SPEN interaction.

BACKGROUND: The RNA recognition motif (RRM) is primarily engaged in the processing of mRNA and rRNA following gene transcription as well as the regulation of RNA transport; it is critical in preserving RNA stability.
RESULTS: In this study, we identified 102 members of the RRM1 gene family in Brassica rapa, which were dispersed across 10 chromosomes with the ninth chromosome being the most extensively distributed. The RRM1 gene family members of Brassica rapa and Arabidopsis thaliana were grouped into 14 subclades (I-XIV) using phylogenetic analysis. Moreover, the results of transcriptome analysis and RT-qPCR indicated that the expression of Brapa05T000840 was upregulated in the cultivars \'Longyou 7\' and \'Longyou 99\' following exposure to cold stress at a temperature of 4 °C for 24 h. The levels of expression in the leaves and growth cones of the \'Longyou 7\' variety were found to be significantly higher than those observed in the \'Longyou 99\' variety under conditions of low temperature and NaCl stress. It illustrates the involvement of the RRM1 gene in the physiological response to both low temperature and salt stress. In addition, it was observed that the survival rate of transgenic BrRBP (Brapa05T000840) Arabidopsis thaliana plants was notably higher compared to that of wild-type plants when subjected to varying durations of low temperature treatment. Furthermore, the expression of the BrRBP gene in transgenic plants exhibited an upward trend as the duration of low temperature treatment increased, reaching its peak at 24 h. The in-vivo enzymatic activity of reactive oxygen species-scavenging enzymes were found to be significantly elevated in comparison to wild-type plants, suggesting that the BrRBP gene may enhance the cold tolerance of Arabidopsis thaliana.
CONCLUSIONS: This study offers a significant foundation for comprehending the regulation mechanism of the RRM1 gene family in winter Brassica rapa subjected to cold stress, as well as for finding key genes associated with cold resistance.

In Saccharomyces cerevisiae, the CBC-Tif4631p-dependent exosomal targeting (CTEXT) complex consisting of Cbc1/2p, Tif4631p and Upf3p promotes the exosomal degradation of aberrantly long 3\'-extended, export-defective transcripts and a small group of normal (termed \'special\') mRNAs. We carried out a systematic analysis of all previously characterized functional domains of the major CTEXT component Tif4631p by deleting each of them and interrogating their involvement in the nuclear surveillance of abnormally long 3\'-extended and export-defective messages. Our analyses show that the N-terminal RNA recognition motif 1 (RRM1) and poly(A)-binding protein (PAB) domains of Tif4631p, spanning amino acid residues, 1-82 and 188-299 in its primary structure, respectively, play a crucial role in degrading these aberrant messages. Furthermore, the physical association of the nuclear exosome with the altered/variant CTEXT complex harboring any of the mutant Tif4631p proteins lacking either the RRM1 or PAB domain becomes abolished. This finding indicates that the association between CTEXT and the exosome is accomplished via interaction between these Tif4631p domains with the major exosome component, Rrp6p. Abolition of interaction between altered CTEXT (harboring any of the RRM1/PAB-deleted versions of Tif4631p) and the exosome further leads to the impaired recruitment of the RNA targets to the Rrp6p subunit of the exosome carried out by the RRM1/PAB domains of Tif4631p. When analyzing the Tif4631p-interacting proteins, we identified a DEAD-box RNA helicase (Dbp2p), as an interacting partner that turned out to be a previously unknown component of CTEXT. The present study provides a more complete description of the CTEXT complex and offers insight into the functional relationship of this complex with the nuclear exosome.

Fused in sarcoma (FUS) is an abundant RNA-binding protein, which drives phase separation of cellular condensates and plays multiple roles in RNA regulation. The RNA-binding ability of FUS protein is crucial to its cellular function. Here, our molecular simulation study on the FUS-RNA complex provides atomic resolution insights into the observations from biochemical studies and also illuminates our understanding of molecular driving forces that mediate the structure, stability, and interaction of the RNA recognition motif (RRM) and RGG domains of FUS with a stem-loop junction RNA. We observe clear cooperativity and division of labor among the ordered (RRM) and disordered domains (RGG1 and RGG2) of FUS that leads to an organized and tighter RNA binding. Irrespective of the length of RGG2, the RGG2-RNA interaction is confined to the stem-loop junction and the proximal stem regions. On the other hand, the RGG1 interactions are primarily with the longer RNA stem. We find that the C terminus of RRM, which make up the \"boundary residues\" that connect the folded RRM with the long disordered RGG2 stretch of the protein, plays a critical role in FUS-RNA binding. Our study provides high-resolution molecular insights into the FUS-RNA interactions and forms the basis for understanding the molecular origins of full-length FUS interaction with RNA.

PARP-1 activation at DNA damage sites leads to the synthesis of long poly(ADP-ribose) (PAR) chains, which serve as a signal for DNA repair. Here we show that FUS, an RNA-binding protein, is specifically directed to PAR through its RNA recognition motif (RRM) to increase PAR synthesis by PARP-1 in HeLa cells after genotoxic stress. Using a structural approach, we also identify specific residues located in the FUS RRM, which can be PARylated by PARP-1 to control the level of PAR synthesis. Based on the results of this work, we propose a model in which, following a transcriptional arrest that releases FUS from nascent mRNA, FUS can be recruited by PARP-1 activated by DNA damage to stimulate PAR synthesis. We anticipate that this model offers new perspectives to understand the role of FET proteins in cancers and in certain neurodegenerative diseases such as amyotrophic lateral sclerosis.