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Abstract We report that in humans, mice, fruit flies, and worms, the ribosomal RNAs and the transcribed spacers of 45S are densely packed with organism-specific sequence motifs that are primarily shared with nervous system genes. The human ribosomal RNAs and 45S spacers contain 1,723 such motifs. Specific combinations of these motifs are predominantly found in 3,430 human nervous system genes, of which 1,046 are genes associated with brain disorders, including autism spectrum disorder and schizophrenia. The sequences of the 1,723 motifs and their locations in the introns and exons of nervous system genes are unique to primates. Experimental evidence indicates that the motifs are contact points for ribosomal-RNA|messenger-RNA and messenger-RNA|messenger-RNA heteroduplexes, present in the binding sites of many RNA-binding proteins, and carried by endogenous small noncoding RNAs. Moreover, several of the motifs’ intergenic copies overlap genome-wide association-study–determined polymorphisms associated with brain disorders and other conditions. Lastly, based on expression data from bulk brain samples from autism spectrum disorder patients and controls, specific combinations of these motifs are enriched only among genes that are downregulated in the patients and only in the cortical areas that are responsible for language, hearing, and vision. This study shows that genomic architecture, the sequences of ribosomal RNAs/spacers, and the sequences of nervous system genes have remained interlocked across 600 million years of evolution through organism-specific motifs. The findings suggest an extensive regulatory layer and can aid in developing new molecular diagnostics and treatments for disorders considered typically human.

Ribosomal RNAs, the most abundant cellular RNA species, have evolved as the structural scaffold and the catalytic center of protein synthesis in every living organism. In eukaryotes, they are produced from a long primary transcript through an intricate sequence of processing steps that include RNA cleavage and folding and nucleotide modification. The mechanisms underlying this process in human cells have long been investigated, but technological advances have accelerated their study in the past decade. In addition, the association of congenital diseases to defects in ribosome synthesis has highlighted the central place of ribosomal RNA maturation in cell physiology regulation and broadened the interest in these mechanisms. Here, we give an overview of the current knowledge of pre-ribosomal RNA processing in human cells in light of recent progress and discuss how dysfunction of this pathway may contribute to the physiopathology of congenital diseases.

Small noncoding RNAs of different origins and classes play several roles in the regulation of gene expression. Here, we show that diverged and rearranged fragments of rDNA units are scattered throughout the human genome and that endogenous small noncoding RNAs are processed by the Microprocessor complex from specific regions of ribosomal RNAs shaping hairpins. These small RNAs correspond to particular sites inside the fragments of rDNA that mostly reside in intergenic regions or the introns of about 1500 genes. The targets of these small ribosomal RNAs (srRNAs) are characterized by a set of epigenetic marks, binding sites of Pol II, RAD21, CBP, and P300, DNase I hypersensitive sites, and by enrichment or depletion of active histone marks. In HEK293T cells, genes that are targeted by srRNAs (srRNA target genes) are involved in differentiation and development. srRNA target genes are enriched with more actively transcribed genes. Our data suggest that remnants of rDNA sequences and srRNAs may be involved in the upregulation or downregulation of a specific set of genes in human cells. These results have implications for diverse fields, including epigenetics and gene therapy.

Osteoarthritis (OA) is a prevalent chronic disease, characterized by progressive joint degeneration and primarily affects older adults. OA leads to reduced functional abilities, a lower quality of life, and an increased mortality rate. Currently, effective treatment options for OA are lacking. Non-coding RNAs (ncRNAs) are functional RNAs transcribed from DNA but not translated into proteins. Among ncRNAs, small interfering RNAs (siRNAs), transfer RNAs (tRNAs), and ribosomal RNAs (rRNAs) have become significant in the field, which is intricately linked to the progression of OA and perform significant regulatory functions in transcription, post-transcription, and post-translation, making them potential biological targets for the prevention, diagnosis, and treatment of OA. This review summarizes the general functions of siRNAs, tRNAs, and rRNAs and their application in OA. The primary focus has been on regulating cartilage degradation. Other participations include regulating synovium, protecting anterior cruciate ligament cells, and diagnosis. No clinical trials were found as challenges such as effective delivery systems, immune responses, long-term effects, and interactions between therapies need to be demonstrated first.

RNA molecules are composed of modular architectural units that define their unique structural and functional properties. Characterization of these building blocks can help interpret RNA structure/function relationships. We present an RNA secondary structure motif and submotif library using dual graph representation and partitioning. Dual graphs represent RNA helices as vertices and loops as edges. Unlike tree graphs, dual graphs can represent RNA pseudoknots (intertwined base pairs). For a representative set of RNA structures, we construct dual graphs from their secondary structures, and apply our partitioning algorithm to identify non-separable subgraphs (or blocks) without breaking pseudoknots. We report 56 subgraph blocks up to nine vertices; among them, 22 are frequently occurring, 15 of which contain pseudoknots. We then catalog atomic fragments corresponding to the subgraph blocks to define a library of building blocks that can be used for RNA design, which we call RAG-3Dual, as we have done for tree graphs. As an application, we analyze the distribution of these subgraph blocks within ribosomal RNAs of various prokaryotic and eukaryotic species to identify common subgraphs and possible ancestry relationships. Other applications of dual graph partitioning and motif library can be envisioned for RNA structure analysis and design.

The heterogeneity of ribosomes, characterized by structural variations, arises from differences in types, numbers, and/or post-translational modifications of participating ribosomal proteins (RPs), ribosomal RNAs (rRNAs) sequence variants plus post-transcriptional modifications, and additional molecules essential for forming a translational machinery. The ribosomal heterogeneity within an individual organism or a single cell leads to preferential translations of selected messenger RNA (mRNA) transcripts over others, especially in response to environmental cues. The role of ribosomal heterogeneity in SARS-CoV-2 coronavirus infection, propagation, related symptoms, or vaccine responses is not known, and a technique to examine these has not yet been developed. Tools to detect ribosomal heterogeneity or to profile translating mRNAs independently cannot identify unique or specialized ribosome(s) along with corresponding mRNA substrate(s). Concurrent characterizations of RPs and/or rRNAs with mRNA substrate from a single ribosome would be critical to decipher the putative role of ribosomal heterogeneity in the COVID-19 disease, caused by the SARS-CoV-2, which hijacks the host ribosome to preferentially translate its RNA genome. Such a protocol should be able to provide a high-throughput screening of clinical samples in a large population that would reach a statistical power for determining the impact of a specialized ribosome to specific characteristics of the disease. These characteristics may include host susceptibility, viral infectivity and transmissibility, severity of symptoms, antiviral treatment responses, and vaccine immunogenicity including its side effect and efficacy. In this study, several state-of-the-art techniques, in particular, chemical probing of ribosomal components or rRNA structures, proximity ligation to generate rRNA-mRNA chimeras for sequencing, nanopore gating of individual ribosomes, nanopore RNA sequencing and/or structural analyses, single-ribosome mass spectrometry, and microfluidic droplets for separating ribosomes or indexing rRNAs/mRNAs, are discussed. The key elements for further improvement and proper integration of the above techniques to potentially arrive at a high-throughput protocol for examining individual ribosomes and their mRNA substrates in a clinical setting are also presented.

The ATP-dependent Clp protease has been well-characterized in Escherichia coli, but knowledge of its function in higher plants is limited. In bacteria, this two-component protease consists of a Ser-type endopeptidase ClpP, which relies on the ATP-dependent unfolding activity from an Hsp100 molecular chaperone to initiate protein degradation. In the chloroplasts of higher plants, multiple isoforms of the proteolytic subunit exist, with Arabidopsis having five ClpPs and four ClpP-like proteins termed ClpR predicted in its genome. In this work we characterized an Arabidopsis mutant impaired in one subunit of the chloroplast-localized Clp protease core, ClpR1. clpR1-1, a virescent mutant, carries a pre-mature stop codon in the clpR1 gene, resulting in no detectable ClpR1 protein. The accumulation of several chloroplast proteins, as well as most of the chloroplast-localized Clp protease subunits, is inhibited in clpR1-1. Unexpectedly, some plastid-encoded proteins do not accumulate, although their transcripts accumulate to wild-type levels. Maturation of 23S and 4.5S chloroplast ribosomal RNA (cp-rRNA) is delayed in clpR1-1, and both RNAs accumulate as higher molecular weight precursors. Also, chloroplasts in clpR1-1 are smaller than in wild type and have fewer thylakoid membranes with smaller grana stacks. We propose that a ClpR1-containing activity is required for chloroplast development and differentiation and in its absence both are delayed.