RNA最新文献

IFITs (interferon-induced proteins with tetratricopeptide repeats) are components of the innate immune response that bind to viral and cellular RNA targets to inhibit translation and replication. The RNA target recognition is guided by molecular patterns, particularly at the RNA 5' ends. IFIT1 preferably binds RNAs modified with the m⁷G cap-0 structure, while RNAs with cap-1 structure are recognized with lower affinity. Less is known about the propensity of IFIT1 to recognize noncanonical RNA 5' ends, including hypermethylated and noncanonical RNA caps. Further insights into the structure-function relationship for IFIT1-RNA interactions are needed but require robust analytical methods. Here, we report a biophysical assay for quick, direct, in-solution affinity assessment of differently capped RNAs with IFIT1. The procedure, which relies on measuring microscale thermophoresis of fluorescently labeled protein as a function of increasing ligand concentration, is applicable to RNAs of various lengths and sequences without the need for their labeling or affinity tagging. Using the assay, we examined 13 canonically and noncanonically 5'-capped RNAs, revealing new binding preferences of IFIT1. The 5' terminal m⁶A mark in the m⁷G cap had a protective function against IFIT1, which was additive with the effect observed for the 2'-O position (m⁶A_m cap-1). In contrast, an increased affinity for IFIT1 was observed for several noncanonical caps, including trimethylguanosine, unmethylated (G), and flavin-adenine dinucleotide caps. The results suggest new potential cellular targets of IFIT1 and may contribute to broadening the knowledge of the innate immune response mechanisms and the more effective design of chemically modified mRNAs.

Respiration in eukaryotes depends on mitochondrial protein synthesis, which is performed by organelle-specific ribosomes translating organelle-encoded mRNAs. Although RNA maturation and stability are central events controlling mitochondrial gene expression, many of the molecular details in this pathway remain elusive. These include cis- and trans-regulatory factors that generate and protect the 3' ends. Here, we mapped the 3' ends of mitochondrial mRNAs of yeasts classified into multiple families of the subphylum Saccharomycotina. We found that the processing of mitochondrial 15S rRNA and mRNAs involves species-specific sequence elements, which we term 3'-end RNA processing elements (3'-RPEs). In Saccharomyces cerevisiae, the 3'-RPE has long been recognized as a conserved dodecamer sequence, which recent studies have shown specifically interacts with the nuclear genome-encoded pentatricopeptide repeat protein Rmd9. We also demonstrate that, analogous to Rmd9 in S. cerevisiae, two Rmd9 orthologs from the Debaryomycetaceae family interact with their respective 3'-RPEs found in mRNAs and 15S rRNA. Thus, Rmd9-dependent processing of mitochondrial RNA precursors may be a common mechanism among the families of the Saccharomycotina subphylum. Surprisingly, we observed that 3'-RPEs often occur upstream of stop codons in complex I subunit mRNAs from yeasts of the CUG-Ser1 clade. We examined two of these mature mRNAs and found that their stop codons are indeed removed. Thus, translation of these stop-codon-less transcripts would require a noncanonical termination mechanism. Our findings highlight Rmd9 as a key evolutionarily conserved factor in both mitochondrial mRNA metabolism and mitoribosome biogenesis in a variety of yeasts.

The entire RNA life cycle, spanning from transcription to decay, is intricately regulated by RNA-binding proteins (RBPs). To understand their precise functions, it is crucial to identify direct targets, pinpoint their exact binding sites, and unravel the underlying specificity in vivo. Individual-nucleotide resolution UV cross-linking and immunoprecipitation 2 (iCLIP2) is a state-of-the-art technique that enables the identification of RBP-binding sites at single-nucleotide resolution. However, in the field of microbiology, optimized iCLIP protocols compared to mammalian systems are lacking. Here, we present the first microbial iCLIP2 approach using the multi-RRM domain protein Rrm4 from the fungus Ustilago maydis as an example. Key challenges, such as inherently high RNase and protease activity in fungi, were addressed by improving mechanical cell disruption and lysis buffer composition. Our modifications increased the yield of cross-link events and improved the identification of Rrm4-binding sites. Thereby, we were able to pinpoint that Rrm4 binds the stop codons of nuclear-encoded mRNAs of mitochondrial respiratory complexes I, III, and V-revealing an intimate link between endosomal mRNA transport and mitochondrial physiology. Thus, our study using U. maydis as an example might serve as a blueprint for optimizing iCLIP2 procedures in other microorganisms with high RNase/protease conditions.

Targeting and manipulating endogenous RNAs in a sequence-specific manner is essential for both understanding RNA biology and developing RNA-targeting therapeutics. RNA-binding zinc fingers (ZnFs) are excellent candidates as designer proteins to expand the RNA-targeting toolbox, due to their compact size and modular sequence recognition. Currently, little is known about how the sequence of RNA-binding ZnF domains governs their binding site specificity. Here, we systematically introduced mutations at the RNA-contacting residues of a well-characterized RNA-binding ZnF protein, ZRANB2, and measured RNA binding of mutant ZnFs using a modified RNA bind-n-seq assay. We identified mutant ZnFs with an altered sequence specificity, preferring to bind a GGG motif instead of the GGU preferred by wild-type ZRANB2. Further, through a series of all-atom molecular dynamics simulations with ZRANB2 and RNA, we characterized changes in the hydrogen-bond network between the protein and RNA that underlie the observed sequence specificity changes. Our analysis of ZRANB2-RNA interactions both in vitro and in silico expands the understanding of ZnF-RNA recognition rules and serves as a foundation for eventual use of RNA-binding ZnFs for programmable RNA targeting.

Uridine insertion/deletion (U-indel) RNA editing of mitochondrial transcripts is a posttranscriptional modification in kinetoplastid organisms, resulting in the generation of mature mRNAs from cryptic precursors. This RNA editing process involves a multiprotein complex holoenzyme and multiple accessory factors. Recent investigations have highlighted the pivotal involvement of accessory RNA-binding proteins (RBPs) in modulating RNA editing in Trypanosoma brucei, often in a transcript-specific manner. DRBD18 is a multifunctional RBP that reportedly impacts the stability, processing, export, and translation of nuclear-encoded mRNAs. However, mass spectrometry studies report DRBD18-RESC interactions, prompting us to investigate its role in mitochondrial U-indel RNA editing. In this study, we demonstrate the specific and RNase-sensitive interaction of DRBD18 with multiple RESC factors. Depletion of DRBD18 through RNA interference in procyclic form T. brucei leads to a significant reduction in the levels of edited A6 and COIII mitochondrial transcripts, whereas its overexpression causes a notable increase in the abundance of these edited mRNAs. RNA immunoprecipitation/qRT-PCR analysis indicates a direct role for DRBD18 in A6 and COIII mRNA editing. We also examined the impact of arginine methylation of DRBD18 in the editing process, revealing that the hypomethylated form of DRBD18, rather than the arginine-methylated version, is essential for promoting these editing events. In conclusion, our findings demonstrate that DRBD18 directly affects the editing of A6 and COIII mRNAs, with its function being modulated by its arginine methylation status, marking the first report of a mitochondrial function for this protein and identifying it as a newly characterized RNA editing auxiliary factor.

Sequencing RNAs that are biologically processed or degraded to less than ∼100 nt typically involves multistep, low-yield protocols with bias and information loss inherent to ligation and/or polynucleotide tailing. We recently introduced ordered two-template relay (OTTR), a method that captures obligatorily end-to-end sequences of input molecules and, in the same reverse transcription step, also appends 5' and 3' sequencing adapters of choice. OTTR has been thoroughly benchmarked for optimal production of microRNA, tRNA and tRNA fragments, and ribosome-protected mRNA footprint libraries. Here we sought to characterize, quantify, and ameliorate any remaining bias or imprecision in the end-to-end capture of RNA sequences. We introduce new metrics for the evaluation of sequence capture and use them to optimize reaction buffers, reverse transcriptase sequence, adapter oligonucleotides, and overall workflow. Modifications of the reverse transcriptase and adapter oligonucleotides increased the 3' and 5' end-precision of sequence capture and minimized overall library bias. Improvements in recombinant expression and purification of the truncated Bombyx mori R2 reverse transcriptase used in OTTR reduced nonproductive sequencing reads by minimizing bacterial nucleic acids that compete with low-input RNA molecules for cDNA synthesis, such that with miRNA input of 3 pg (<1 fmol), fewer than 10% of sequencing reads are bacterial nucleic acid contaminants. We also introduce a rapid, automation-compatible OTTR protocol that enables gel-free, length-agnostic enrichment of cDNA duplexes from unwanted adapter-only side products. Overall, this work informs considerations for unbiased end-to-end capture and annotation of RNAs independent of their sequence, structure, or posttranscriptional modifications.

Prominent members of the ribosomal RNA adenine dimethylase (RRAD) family of enzymes facilitate ribosome maturation by dimethylating 2 nt of small subunit rRNA, including the human DIMT1 and bacterial KsgA enzymes. A subgroup of RRAD enzymes, named erythromycin resistance methyltransferases (Erm), dimethylate a specific nucleotide in large subunit rRNA to confer antibiotic resistance. How these enzymes regulate methylation so that it only occurs on the specific substrate is not fully understood. While performing random mutagenesis on the catalytic domain of ErmE, we discovered that mutants in an N-terminal region of the protein that is disordered in the ErmE crystal structure are associated with a loss of antibiotic resistance. By subjecting site-directed mutants of ErmE and KsgA to phenotypic and in vitro assays, we found that the N-terminal region is critical for activity in RRAD enzymes: The N-terminal basic region promotes rRNA binding, and the conserved motif likely assists in juxtaposing the adenosine substrate and the S-adenosylmethionine cofactor. Our results and emerging structural data suggest that this dynamic, N-terminal region of RRAD enzymes becomes ordered upon rRNA binding, forming a cap on the active site required for methylation.

Structures in the 5' untranslated regions (UTRs) of mRNAs can physically modulate translation efficiency by impeding the scanning ribosome or by sequestering the translational start site. We assessed the impact of stable protein binding in 5'- and 3'-UTRs on translation efficiency by targeting the MS2 coat protein to a reporter RNA via its hairpin recognition site. Translation was assessed from the reporter RNA when coexpressed with MS2 coat proteins of varying affinities for the RNA, and at different expression levels. Binding of high-affinity proteins in the 5'-UTR hindered translation, whereas no effect was observed when the coat protein was targeted to the 3'-UTR. Inhibition of translation increased with coat protein concentration and affinity, reaching a maximum of 50%-70%. MS2 proteins engineered to bind two reporter mRNA sites had a stronger effect than those binding a single site. Our findings demonstrate that protein binding in an mRNA 5'-UTR physically impedes translation, with the effect governed by affinity, concentration, and sterics.

The international symposium ASOBIOTICS 2024 brought together scientists across disciplines to discuss the challenges of advancing antibacterial antisense oligomers (ASOs) from basic research to clinical application. Hosted by the Helmholtz Institute for RNA-based Infection Research (HIRI) in Wurzburg, Germany, on September 12-13th, 2024, the event featured presentations covering major milestones and current challenges of this antimicrobial technology and its applications against pathogens, commensals, and bacterial viruses. General design principles and modification of ASOs based on peptide nucleic acid (PNA) or phosphorodiamidate-morpholino-oligomer (PMO) chemistry, promising cellular RNA targets, new delivery technologies, as well as putative resistance mechanisms were discussed. A panel discussion noted the challenge of nomenclature: antibacterial ASOs lack a single, universally used name. To address this, the term "asobiotics" was proposed to unite a community of like-minded scientists that are committed to advancing ASOs as antimicrobials. A consistent name will simplify literature searches and help scientists and funders appreciate the potential of programmable RNA antibiotics to combat antimicrobial resistance and enable precise microbiome editing.

Messenger RNA (mRNA) translational control plays a pivotal role in regulating cellular proteostasis under physiological and pathological conditions. Dysregulated mRNA translation is pervasive in cancer, in which protein synthesis is elevated to support accelerated cell growth and proliferation. Consequently, targeting the mRNA translation machinery has emerged as a therapeutic strategy to treat cancer. In this perspective, we summarize the current knowledge of translation dysregulation in cancer, with emphasis on the eukaryotic translation initiation factor 4F (eIF4F) complex. We outline recent endeavors to apply this knowledge to develop novel treatment strategies to combat cancer.