🧬 PubMed RNase III Daily Digest

Bacterial ribonucleases (RNases) are central components of post-transcriptional networks underlying environmental adaptation. However, their contribution to the ecological specialization of bacteria with complex lifestyles, such as nitrogen-fixing legume symbionts, remains poorly understood. Here, we investigated the role of the double-stranded RNase III ortholog (SmRNase III) in Sinorhizobium meliloti, the symbiotic partner of alfalfa (Medicago sativa L.). Loss of SmRNase III function affected the expression of nearly 30% of protein-coding genes and 12% of annotated non-coding RNAs (sRNAs). Remarkably, more than 70% of these changes occurred under the microaerobic conditions typical of symbiotic nodules. Many SmRNase III-dependent transcripts encode pathways supporting microaerobic metabolism and nitrogen fixation in endosymbiotic bacteroids. Analysis of sequencing read coverage identified putative consensus cleavage signatures enriched in mRNA 5' untranslated regions, suggesting preferential processing at these sites. Altered expression of sRNAs and/or their predicted mRNA targets further supports a role for SmRNase III in sRNA-mediated silencing. Consistently, in vitro assays showed that base‑pairing between nifK (encoding the β‑subunit of the nitrogenase MoFe protein) and the antisense RNA asNifK promotes SmRNase III-mediated cleavage. In vivo assays further supported that silencing of nifK and dctA (encoding a major dicarboxylate transporter) requires SmRNase III, with dctA regulation involving a base‑pairing interaction between the trans‑sRNA AbcR1 and a predicted SmRNase III cleavage site within the mRNA. Our findings reveal a major impact of SmRNase III on shaping the symbiotic transcriptome of S. meliloti and provide a foundation for deeper investigation into RNase III-mediated regulation in rhizobia.

Chloroplast ribosome biogenesis is essential for plastid development, yet the regulatory mechanisms underlying chloroplast rRNA maturation remain incompletely understood. Here, we characterize the role of DEFECTIVE CHLOROPLASTS AND LEAVES (DCL) in chloroplast rRNA processing in Arabidopsis thaliana. DCL is conserved in photosynthetic eukaryotes and localizes to chloroplasts, whereas its paralogs exhibit distinct localization patterns including chloroplast-mitochondrial dual localization and nuclear-specific targeting. Loss of DCL results in seedling lethality and an ivory leaf phenotype, accompanied by severely decreased accumulation of chloroplast ribosomes. RNA blotting and circularized RT-PCR analyses reveal that DCL is required for the accurate processing of the 16S rRNA and the 23S-4.5S dicistronic precursor as well as for the proper 'hidden break' processing of 23S rRNA. Yeast two-hybrid assays demonstrate that DCL physically interacts with three key factors involved in chloroplast rRNA processing including the mini-RNase III-like enzymes RNC3/RNC4, ribonuclease RNase J (RNJ), and endonuclease YbeY. Thus, our results indicate that DCL is a critical eukaryotic-specific factor that coordinates early endonucleolytic cleavage steps during chloroplast rRNA maturation, thereby enabling proper ribosome assembly and chloroplast translation.

Spatiotemporal regulation of Dicer is essential for small RNA biogenesis and fertility, yet how its helicase domain is controlled remains unclear. Using Caenorhabditis elegans, we identify a regulatory role for the arginine-rich GRARR motif within helicase domain motif VI of DCR-1. Mutating conserved arginines in this sequence disrupts maternal 26 G endo-siRNA production, impairs oocyte meiosis I and II, and reduces fertility. Biochemically, an asymmetrically dimethylated DCR-1 GRA[R495*]R peptide enhances interaction with ERI-5, a tandem-Tudor protein in the ERIC complex, while loss of DCR-1(R495) diminishes this interaction in vivo. Genetically, eri-5 deletion phenocopies the dcr-1 R495K mutant, supporting a functional partnership in 26 G siRNA biogenesis. Notably, these defects parallel those seen in DCR-1 phosphorylation mutants in the catalytic domain. AlphaFold modeling suggests that arginine methylation in the helicase domain and serine phosphorylation in catalytic domain may operate in a coordinated manner to modulate DCR-1 conformation, effector recruitment, and proper execution of the oocyte meiotic program.

mRNA-based therapeutics are commonly produced through T7 RNA polymerase-mediated in vitro transcription. Introducing these exogenous RNAs into human cells activates an RNA sensor protein kinase R (PKR), which suppresses translation initiation and reduces their therapeutic effectiveness. Incorporating uridine analogs into these transcripts prevents PKR activation and translation shutdown, but the underlying mechanism remains unclear. Here, we demonstrate that treating T7 RNA polymerase-produced transcripts with RNase III, which selectively degrades double-stranded RNA (dsRNA), blocks PKR activation and downstream translation-inhibition events, including eIF2α phosphorylation and stress granule formation in human cells. Interestingly, dsRNAs generated with uridine analogs robustly induce eIF2α phosphorylation and stress granules to the same extent as dsRNA containing uridine. These findings indicate that uridine analogs do not prevent PKR from detecting dsRNA. Instead, we show that uridine analogs decrease the production of T7 RNA polymerase byproducts, including antisense RNA and dsRNA, which activate PKR and downstream stress responses. Finally, we demonstrate that higher amounts of exogenous RNA, lacking T7 RNA polymerase byproducts, can induce stress granules independently of PKR and phospho-eIF2α, but dependent on stress granule scaffold proteins G3BP1 and G3BP2. Together, our findings show that uridine analogs mitigate PKR signaling not by blocking mRNA-PKR interactions but by minimizing dsRNA byproducts from T7 polymerase transcription. Furthermore, stress granule formation in response to high levels of exogenous RNA can occur through a mechanism that does not depend on PKR but relies on G3BP1 and G3BP2. These insights clarify the role of uridine analogs in PKR activation and may inform future therapeutic RNA design.

ER stress underlies numerous severe pathologies. We have metabolically perturbed normal fibroblasts to study the biological roles of microRNAs (miRs) under mild and extended ER stress. We now report that miR-4488 quenches inflammation-associated gene expression in such metabolically perturbed cells. Remarkably, generation of miR-4488 is Drosha-independent. Furthermore, we define miR-4488 as a noncanonical miRNA derived from the expansion segment ES7L of the 28S ribosomal RNA. Moreover, its generation involves the autophagy-lysosome route and is inhibited when this pathway is blocked, thus unveiling an anti-inflammatory role for ribosomal RNA and lysosomes, engaged at the onset of stress. Mechanistically, miR-4488 suppresses the expression of NFKB2 and RELB, whose mRNAs specifically associate with miR-4488 exclusively upon stress. This selectivity suggests that miR-4488 may bear promise for treating mild ER stress-associated diseases.