Discovery, structure, mechanisms, and evolution of protein-only RNase P enzymes.

The endoribonuclease RNase P is responsible for tRNA 5' maturation in all domains of life. A unique feature of RNase P is the variety of enzyme architectures, ranging from dual- to multi-subunit ribonucleoprotein forms with catalytic RNA subunits to protein-only enzymes, the latter occurring as single- or multi-subunit forms or homo-oligomeric assemblies. The protein-only enzymes evolved twice: a eukaryal protein-only RNase P termed PRORP and a bacterial/archaeal variant termed homolog of Aquifex RNase P (HARP); the latter replaced the RNA-based enzyme in a small group of thermophilic bacteria but otherwise coexists with the ribonucleoprotein enzyme in a few other bacteria as well as in those archaea that also encode a HARP. Here we summarize the history of the discovery of protein-only RNase P enzymes and review the state of knowledge on structure and function of bacterial HARPs and eukaryal PRORPs, including human mitochondrial RNase P as a paradigm of multi-subunit PRORPs. We also describe the phylogenetic distribution and evolution of PRORPs, as well as possible reasons for the spread of PRORPs in the eukaryal tree and for the recruitment of two additional protein subunits to metazoan mitochondrial PRORP. We outline potential applications of PRORPs in plant biotechnology and address diseases associated with mutations in human mitochondrial RNase P genes. Finally, we consider possible causes underlying the displacement of the ancient RNA enzyme by a protein-only enzyme in a small group of bacteria.

P finder: genomic and metagenomic annotation of RNase P RNA gene (rnpB).

BACKGROUND: The rnpB gene encodes for an essential catalytic RNA (RNase P). Like other essential RNAs, RNase P's sequence is highly variable. However, unlike other essential RNAs (i.e. tRNA, 16 S, 6 S,...) its structure is also variable with at least 5 distinct structure types observed in prokaryotes. This structural variability makes it labor intensive and challenging to create and maintain covariance models for the detection of RNase P RNA in genomic and metagenomic sequences. The lack of a facile and rapid annotation algorithm has led to the rnpB gene being the most grossly under annotated essential gene in completed prokaryotic genomes with only a 24% annotation rate. Here we describe the coupling of the largest RNase P RNA database with the local alignment scoring algorithm to create the most sensitive and rapid prokaryote rnpB gene identification and annotation algorithm to date. RESULTS: Of the 2772 completed microbial genomes downloaded from GenBank only 665 genomes had an annotated rnpB gene. We applied P Finder to these genomes and were able to identify 2733 or nearly 99% of the 2772 microbial genomes examined. From these results four new rnpB genes that encode the minimal T-type P RNase P RNAs were identified computationally for the first time. In addition, only the second C-type RNase P RNA was identified in Sphaerobacter thermophilus. Of special note, no RNase P RNAs were detected in several obligate endosymbionts of sap sucking insects suggesting a novel evolutionary adaptation. CONCLUSIONS: The coupling of the largest RNase P RNA database and associated structure class identification with the P Finder algorithm is both sensitive and rapid, yielding high quality results to aid researchers annotating either genomic or metagenomic data. It is the only algorithm to date that can identify challenging RNAse P classes such as C-type and the minimal T-type RNase P RNAs. P Finder is written in C# and has a user-friendly GUI that can run on multiple 64-bit windows platforms (Windows Vista/7/8/10). P Finder is free available for download at https://github.com/JChristopherEllis/P-Finder as well as a small sample RNase P RNA file for testing.

Essential is not irreplaceable: fitness dynamics of experimental E. coli RNase P RNA heterologous replacement.

While critical cellular components-such as the RNA moiety of bacterial ribonuclease P-can sometimes be replaced with a highly divergent homolog, the cellular response to such perturbations is often unexpectedly complex. RNase P is a ubiquitous and essential ribonucleoprotein involved in the processing of multiple RNA substrates, including tRNAs, small non-coding RNAs and intergenic operons. In Bacteria, RNase P RNAs have been subdivided-based on their secondary and tertiary structures-into two major groups (A and B), each with a distinct phylogenetic distribution. Despite the vast phylogenetic and structural gap that separates the two RNase P RNA classes, previous work suggested their interchangeability. Here, we explore in detail the functional and fitness consequences of replacing the endogenous Type-A Escherichia coli RNase P RNA with a Type-B homolog derived from Bacillus subtilis, and show that E. coli cells forced to survive with a chimeric RNase P as their sole source of RNase P activity exhibit extremely variable responses. The chimeric RNase P alters growth rates-used here as an indirect measure of fitness-in unpredictable ways, ranging from 3- to 20-fold reductions in maximal growth rate. The transcriptional behavior of cells harboring the chimeric RNAse P is also perturbed, affecting the levels of at least 79 different transcripts. Such transcriptional plasticity represents an important mechanism of transient adaptation which, when coupled with the emergence and eventual fixation of compensatory mutations, enables the cells to overcome the disruption of this tightly coevolving ribonucleoprotein.

Naming 'junk': human non-protein coding RNA (ncRNA) gene nomenclature.

Previously, the majority of the human genome was thought to be 'junk' DNA with no functional purpose. Over the past decade, the field of RNA research has rapidly expanded, with a concomitant increase in the number of non-protein coding RNA (ncRNA) genes identified in this 'junk'. Many of the encoded ncRNAs have already been shown to be essential for a variety of vital functions, and this wealth of annotated human ncRNAs requires standardised naming in order to aid effective communication. The HUGO Gene Nomenclature Committee (HGNC) is the only organisation authorised to assign standardised nomenclature to human genes. Of the 30,000 approved gene symbols currently listed in the HGNC database (http://www.genenames.org/search), the majority represent protein-coding genes; however, they also include pseudogenes, phenotypic loci and some genomic features. In recent years the list has also increased to include almost 3,000 named human ncRNA genes. HGNC is actively engaging with the RNA research community in order to provide unique symbols and names for each sequence that encodes an ncRNA. Most of the classical small ncRNA genes have now been provided with a unique nomenclature, and work on naming the long (>200 nucleotides) non-coding RNAs (lncRNAs) is ongoing.

Deciphering RNA structural diversity and systematic phylogeny from microbial metagenomes.

Metagenomics has been employed to systematically sequence, classify, analyze and manipulate the entire genetic material isolated from environmental samples. Finding genes within metagenomic sequences remains a formidable challenge, and noncoding RNA genes other than those encoding rRNA and tRNA are not well annotated in metagenomic projects. In this work, we identify, validate and analyze the genes coding for RNase P RNA (P RNA) from all published metagenomic projects. P RNA is the RNA subunit of a ubiquitous endoribonuclease RNase P that consists of one RNA subunit and one or more protein subunits. The bacterial P RNAs are classified into two types, Type A and Type B, based on the constituents of the structure involved in precursor tRNA binding. Archaeal P RNAs are classified into Type A and Type M, whereas the Type A is ancestral and close to Type A bacterial P RNA. Bacterial and some archaeal P RNAs are catalytically active without protein subunits, capable of cleaving precursor tRNA transcripts to produce their mature 5'-termini. We have found 328 distinctive P RNAs (320 bacterial and 8 archaeal) from all published metagenomics sequences, which led us to expand by 60% the total number of this catalytic RNA from prokaryotes. Surprisingly, all newly identified P RNAs from metagenomics sequences are Type A, i.e. neither Type B bacterial nor Type M archaeal P RNAs are found. We experimentally validate the authenticity of an archaeal P RNA from Sargasso Sea. One of the distinctive features of some new P RNAs is that the P2 stem has kinked nucleotides in its 5' strand. We find that the single nucleotide J2/3 joint region linking the P2 and P3 stem that was used to distinguish a bacterial P RNA from an archaeal one is no longer applicable, i.e. some archaeal P RNAs have only one nucleotide in the J2/3 joint. We also discuss the phylogenetic analysis based on covariance model of P RNA that offers a few advantages over the one based on 16S rRNA.

Inventory and analysis of the protein subunits of the ribonucleases P and MRP provides further evidence of homology between the yeast and human enzymes.

The RNases P and MRP are involved in tRNA and rRNA processing, respectively. Both enzymes in eukaryotes are composed of an RNA molecule and 9-12 protein subunits. Most of the protein subunits are shared between RNases P and MRP. We have here performed a computational analysis of the protein subunits in a broad range of eukaryotic organisms using profile-based searches and phylogenetic methods. A number of novel homologues were identified, giving rise to a more complete inventory of RNase P/MRP proteins. We present evidence of a relationship between fungal Pop8 and the protein subunit families Rpp14/Pop5 as well as between fungal Pop6 and metazoan Rpp25. These relationships further emphasize a structural and functional similarity between the yeast and human P/MRP complexes. We have also identified novel P and MRP RNAs and analysis of all available sequences revealed a K-turn motif in a large number of these RNAs. We suggest that this motif is a binding site for the Pop3/Rpp38 proteins and we discuss other structural features of the RNA subunit and possible relationships to the protein subunit repertoire.

Isolation and molecular evolution of the selenocysteine tRNA (Cf TRSP) and RNase P RNA (Cf RPPH1) genes in the dog family, Canidae.

In an effort to identify rapidly evolving nuclear sequences useful for phylogenetic analyses of closely related species, we isolated two genes transcribed by RNA polymerase III (pol III), the selenocysteine tRNA gene (TRSP) and an RNase P RNA (RPPH1) gene from the domestic dog (Canis familiaris). We focus on genes transcribed by pol III because their coding regions are small (generally 100-300 base pairs [bp]) and their essential promoter elements are located within a couple of hundred bps upstream of the coding region. Therefore, we predicted that regions flanking the coding region and outside of the promoter elements would be free of constraint and would evolve rapidly. We amplified TRSP from 23 canids and RPPH1 from 12 canids and analyzed the molecular evolution of these genes and their utility as phylogenetic markers for resolving relationships among species in Canidae. We compared the rate of evolution of the gene-flanking regions to other noncoding regions of nuclear DNA (introns) and to the mitochondrial encoded COII gene. Alignment of TRSP from 23 canids revealed that regions directly adjacent to the coding region display high sequence variability. We discuss this pattern in terms of functional mechanisms of transcription. Although the flanking regions evolve no faster than introns, both genes were found to be useful phylogenetic markers, in part, because of the synapomorphic indels found in the flanking regions. Gene trees generated from the TRSP and RPPH1 loci were generally in agreement with the published mtDNA phylogeny and are the first phylogeny of Canidae based on nuclear sequences.