RGG-motif proteins play a crucial role in determining mRNA fate. Suppressor of clathrin deficiency 6 (Scd6) is a conserved RGG-motif containing RNP condensate-resident, translation repressor, and decapping activator protein in Saccharomyces cerevisiae. Identifying protein factors that can modulate Scd6 function is critical to understanding the regulation of mRNA fate by Scd6. In this study, using an approach that combined mRNA tethering assay with flow cytometry, we screened 50 genes for their role in modulating the translation repression activity of Scd6. We identified eight conserved modulators with human homologs. Of these, we further characterised in detail guanine nucleotide exchange factor (GEF) Rom2 (Rho1 multicopy suppressor) and glycolytic enzyme Tdh3 (Triose phosphate dehydrogenase 3), which, respectively, impede and promote translation repression activity of Scd6. Our study reveals that Rom2 negatively regulates the arginine methylation of Scd6 and antagonises its localisation to P-bodies. Tdh3, on the other hand, promotes Scd6 interaction with Hmt1, thereby promoting the arginine methylation of Scd6 and enhanced eIF4G1 interaction, which is known to promote its repression activity. Identifying these novel modulators provides exciting new insights into the role of a metabolic enzyme of the glycolytic pathway and guanine nucleotide exchange factor implicated in the cell wall integrity pathway in regulating Scd6 function and, thereby, cytoplasmic mRNA fate.

N6-methyladenosine (m6A) is a fundamentally important RNA modification for gene regulation, whose function is achieved through m6A readers. However, whether and how m6A readers play regulatory roles during fruit ripening and quality formation remains unclear. Here, we characterized SlYTH2 as a tomato m6A reader protein and profiled the binding sites of SlYTH2 at the transcriptome-wide level. SlYTH2 undergoes liquid-liquid phase separation and promotes RNA-protein condensate formation. The target mRNAs of SlYTH2, namely m6A-modified SlHPL and SlCCD1B associated with volatile synthesis, are enriched in SlYTH2-induced condensates. Through polysome profiling assays and proteomic analysis, we demonstrate that knockout of SlYTH2 expedites the translation process of SlHPL and SlCCD1B, resulting in augmented production of aroma-associated volatiles. This aroma enrichment significantly increased consumer preferences for CRISPR-edited fruit over wild type. These findings shed light on the underlying mechanisms of m6A in plant RNA metabolism and provided a promising strategy to generate fruits that are more attractive to consumers.

Pumilio (Pum) is an RNA-binding protein and translational repressor important to diverse biological processes. In the Drosophila ovary, Pum is expressed in female germline stem cells (GSCs), wherein it acts as an intrinsic stem cell maintenance factor via repressing target mRNAs that are as yet mostly unknown. Pum recognizes the Pum binding sequence (PBS) in the mRNA 3\'UTR through its C-terminus Puf domain. Translational repression is mediated through its N-terminal domain, but the molecular mechanism remains largely unknown. We previously showed that Bag-of-marbles, a critical differentiation-promoting factor of female GSCs, physically interacts with the N-terminus of Pum. We further showed that this interaction is critical to Bam inhibition of Pum repressive action in cultured cells, but the physiological relevance was not addressed. Here we design an in vivo GFP translational reporter bearing the PBS in its 3\'UTR and show that GFP expression is reduced in cells wherein Pum is known to be active. Furthermore, we demonstrate in pum mutant ovary that this GFP repression requires Pum, and also that the sensor faithfully monitors Pum activity. Finally, we show that forced expression of Bam inhibits Pum-mediated repression, validating that Bam inhibits Pum activity in vivo.

Since the discovery of microRNAs (miRNAs) in Caenorhabditis elegans, our understanding of their cellular function has progressed continuously. Today, we have a good understanding of miRNA-mediated gene regulation, miRNA-mediated cross talk between genes including competing endogenous RNAs, and miRNA-mediated signaling transduction both in normal human physiology and in diseases.Besides, these noncoding RNAs have shown their value for clinical applications, especially in an oncological context. They can be used as reliable biomarkers for cancer diagnosis and prognosis and attract increasing attention as potential therapeutic targets. Many achievements made in the miRNA field are based on joint efforts from computational and molecular biologists. Systems biology approaches, which integrate computational and experimental methods, have played a fundamental role in uncovering the cellular functions of miRNAs.In this chapter, we review and discuss the role of miRNAs in oncology from a system biology perspective. We first describe biological facts about miRNA genetics and function. Next, we discuss the role of miRNAs in cancer progression and review the application of miRNAs in cancer diagnostics and therapy. Finally, we elaborate on the role that miRNAs play in cancer gene regulatory networks. Taken together, we emphasize the importance of systems biology approaches in our continued efforts to study miRNA cancer regulation.

Sex differences are pervasive in human health and disease. One major key to sex-biased differences lies in the sex chromosomes. Although the functions of the X chromosome proteins are well appreciated, how they compare with their Y chromosome homologs remains elusive. Herein, using ensemble and single-molecule techniques, we report that the sex chromosome-encoded RNA helicases DDX3X and DDX3Y are distinct in their propensities for liquid-liquid phase separation (LLPS), dissolution, and translation repression. We demonstrate that the N-terminal intrinsically disordered region of DDX3Y more strongly promotes LLPS than the corresponding region of DDX3X and that the weaker ATPase activity of DDX3Y, compared with DDX3X, contributes to the slower disassembly dynamics of DDX3Y-positive condensates. Interestingly, DDX3Y-dependent LLPS represses mRNA translation and enhances aggregation of FUS more strongly than DDX3X-dependent LLPS. Our study provides a platform for future comparisons of sex chromosome-encoded protein homologs, providing insights into sex differences in RNA metabolism and human disease.

Severe ethanol stress (>9% v/v) induces pronounced translation repression in yeast cells. However, some proteins, which are exceptionally synthesized even under translation repression, play important roles in ethanol tolerance. These proteins are expected to provide important clues for elucidating the survival strategies of yeast cells under severe ethanol stress. In this study, we identified Hsp78 as a protein effectively synthesized under severe ethanol stress. As Hsp78 is involved in mitochondrial protein quality control, we investigated the effect of severe ethanol stress on mitochondrial proteins and found that Ilv2, Kgd1, and Aco1 aggregated with Hsp78 under severe ethanol stress, forming mitochondrial deposition sites for denatured proteins, called DUMPs (Deposits of Unfolded Mitochondrial Proteins). Aggregation of mitochondrial proteins and formation of DUMPs were accelerated in hsp78∆ cells compared with those in wild-type cells. During the recovery process after ethanol removal, aggregated Ilv2 and DUMP levels rapidly decreased in wild-type cells but were maintained for a long time (>180 min) in hsp78Δ cells. Furthermore, the frequency of respiration-deficient mutants caused by severe ethanol stress was higher in hsp78∆ cells than in wild-type cells. These results indicate that severe ethanol stress damaged mitochondrial proteins and that Hsp78 was preferentially synthesized to cope with the damage, thereby suppressing the rapid increase in aggregated protein levels under stress and achieving proper clearance of aggregated proteins during the recovery process. This study provides novel insights into the adverse effects of ethanol on mitochondria and yeast response to severe ethanol stress.

When encountering oxidative stress, organisms selectively upregulate antioxidant genes and simultaneously suppress the translation of most other proteins. Eukaryotes employ multiple strategies to adjust translation at both the initiation and elongation stages; however, how prokaryotes modulate translation under oxidative stress remains unclear. Here, we report that upon hydrogen peroxide (H2O2) challenge, Streptococcus oligofermentans reduced translation via RNase Z (So-RNaseZ) oxidative degradation, thus hindering tRNA maturation. S. oligofermentans encodes all CCA-less tRNAs that require So-RNaseZ for 3\' end maturation. A combination of nonreducing SDS-PAGE and liquid chromatography/tandem mass spectrometry (LC/MS-MS) assays demonstrated that H2O2 oxidation induced Cys38-Cys149 disulfide linkages in recombinant So-RNaseZ protein, and serine substitution of Cys38 or Cys149 abolished these disulfide linkages. Consistently, redox Western blotting also determined intramolecular disulfide-linked So-RNaseZ in H2O2-treated S. oligofermentans cells. The disulfide-linked So-RNaseZ and monomer were both subject to proteolysis, whereas C149S mutation alleviated oxidative degradation of So-RNaseZ, suggesting that H2O2-mediated disulfide linkages substantially contributed to So-RNaseZ degradation. Accordingly, Northern blotting determined that tRNA precursor accumulation and mature tRNA species decrease in H2O2-treated S. oligofermentans. Moreover, reduced overall protein synthesis, as indicated by puromycin incorporation, and retarded growth of S. oligofermentans occurred in an H2O2 concentration-dependent manner. Overexpression of So-RNaseZ not only elevated tRNA precursor processing and protein synthesis but also partly rescued H2O2-suppressed S. oligofermentans growth. Moreover, So-RNaseZ oxidative degradation-mediated translation repression elevated S. oligofermentans survival under high H2O2 stress. Therefore, this work found that So-RNaseZ oxidative degradation-impeded tRNA maturation contributes to streptococcal translation repression and provides the oxidative stress adaptability for S. oligofermentans. IMPORTANCE Translation regulation is a common strategy used by organisms to reduce oxidative damage. Catalase-negative streptococci produce as well as tolerate high levels of H2O2. This work reports a novel translation regulation mechanism employed by Streptococcus oligofermentans in response to H2O2 challenge, in which the key tRNA endonuclease So-RNaseZ is oxidized to form Cys38-Cys149 disulfide linkages and both the disulfide-linked So-RNaseZ and monomers are subject to proteolysis; thus, tRNA maturation, protein translation, and growth are all suppressed. Notably, So-RNaseZ oxidative degradation-mediated translation repression offers oxidative adaptability to S. oligofermentans and enhances its survival against high H2O2 challenge. So-RNaseZ orthologs and H2O2-sensitive cysteines (Cys38 and Cys149) are widely distributed in Streptococcus and Lactococcus species genomes, which also encode all CCA-less tRNAs and lack catalase. Therefore, RNase Z oxidative degradation-based translation regulation could be widely employed by these lactic acid bacteria, including pathogenic streptococci, to cope with H2O2.

Complex cascades of RNA-binding proteins regulate the mRNA metabolism and influence gene expression. Several distinct proteins act at different stages of mRNA life cycle. SR family proteins in yeast are implicated in mRNA processing and nuclear export. In this report, we uncover the role of an SR/RGG-motif containing mRNA export factor Gbp2 in mRNA translation regulation. We demonstrate that Gbp2 localizes to cytoplasmic granules upon heat shock and oxidative stress. Our pull-down assays demonstrate that Gbp2 directly binds to the conserved translation factor eIF4G1 via its RGG motif. We further mapped the region on eIF4G1 to which Gbp2 binds and observed that the binding region overlaps with another translation repressor Sbp1. We found that the RGG-motif deletion mutant is defective in localizing to polysome fractions. Upon tethering Gbp2 to a GFP reporter mRNA in vivo, translation of GFP reporter decreased significantly indicating that Gbp2 acts as a translation repressor. Consistent with these results, we show that Gbp2 can directly repress mRNA translation in the in vitro translation systems in an RGG-motif dependent manner. Taken together, our results establish that the mRNA export factor Gbp2 has a vital role in repressing translation of mRNA. We propose that Gbp2 is a multifaceted RGG-motif protein responsible for translational repression without affecting mRNA levels.

Background: RNA binding proteins play crucial role in determining if a given mRNA will be translated, stored, or degraded. Sbp1 is an RGG-motif containing protein that is implicated in affecting mRNA decapping and translation. Sbp1 represses translation by binding eIF4G1 through its RGG-motif and activates decapping when overexpressed. In this report, we have assessed the genetic interaction of Sbp1 with decapping activators such as Dhh1, Pat1, and Scd6. We have further analyzed the importance of different domains and specific conserved residues of Sbp1 in its ability to cause over-expression mediated growth defect. Method: Sequence alignment was performed to identify conserved aromatic residues to be mutated. Using site-directed mutagenesis several point mutations and domain deletions were created in Sbp1 expressed under a galactose-inducible promoter. The mutants were tested for their ability to cause growth defect upon over-expression. The ability of Sbp1 to affect over-expression mediated growth defect of other decapping activators was tested using growth assay. Live cell imaging was done to study localization of Sbp1 and its RRM-deletion mutants to RNA granules upon glucose starvation. Results: Mutation of several aromatic residues in the RGG-motif and that of the phosphorylation sites in the RRM domain of Sbp1 did not affect the growth defect phenotype. Deletion of another eIF4G1-binding RGG-motif protein Scd6 does not affect the ability of Sbp1 to cause growth defect. Moreover, absence of Sbp1 did not affect the growth defect phenotypes observed upon overexpression of decapping activators Dhh1 and Pat1. Strikingly deletion of both the RRM domains (RRM1 and RRM2) and not the RNP motifs within them compromised the growth defect phenotype. Sbp1 mutant lacking both RRM1 and RRM2 was highly defective in localizing to RNA granules.   Conclusion: This study identifies an important role of RRM domains independent of the RNP motif in Sbp1 function.

Over the past decades, tRNA was found to be a rich hub of RNA modifications such as 1-methyladenosine and 5-methycytosine modifications and others, holding more than half of all modifications occurring in RNA molecules. Moreover, tRNA was discovered to be a source of various small noncoding RNA species, such as the stress induced angiogenin cleaved tRNA halves (tiRNA) or the miRNA like tRNA derived fragments. tRNA cleavage under stress was fist discovered in bacteria and later was found to be conserved across different species, including mammals. Under cellular stress conditions, tRNA undergoes conformational changes and angiogenin cleaves it into 3\' and 5\' halves. 5\'tiRNA halves were shown to repress protein translations. tRNA cleavage is thought of to be a cytoprotective mechanism by which cells evade apoptosis, however some data hints to the opposite; that tiRNA are cytotoxic or at least related to apoptosis initiation. tRNA cleavage also was shown to be affected by tRNA modifications via different enzymes in the cytosol and mitochondria. In this review, we will highlight the biology of tRNA cleavage, show the evidence of it being cytoprotective or a marker of cell death and shed a light on its role in disease models and human diseases as well as possible future directions in this field of RNA research.