RsaOG, a new Staphylococcal family of highly transcribed non-coding RNA
The expression of trans-acting small RNAs in firmicutes has been poorly documented to date. This gap is being filled quickly in the genus Staphylococcus, which is both a model firmicute and an important human pathogen. Here we analyse RsaOG, a novel small RNA family specific to Staphylococcus and highly transcribed. This well conserved element, first discovered in a computational screen, was precisely mapped in the genome by RACE mapping and the identification of a putative transcriptional promoter. The proposed secondary structure presents two highly conserved unpaired sequences, part of which can form a pseudoknot. We suggest a possible involvement of the remaining conserved single stranded region in trans regulatory interactions.
A novel family of plasmid-transferred anti-sense ncRNAs
The genome of Xanthomonas campestris pv. vesicatoria encodes a constitutively expressed small RNA, which we designate PtaRNA1, “Plasmid transferred anti-sense RNA”. It exhibits all hallmarks of a novel RNA antitoxin that proliferates by frequent horizontal transfer. It shows an erratic phylogenetic distribution with occurrences on chromosomes in a few individual strains distributed across both beta- and gamma-proteobacteria. Moreover, a homologous gene located on plasmid pMATVIM-7 of Pseudomonas aeruginosa is found. All ptaRNA1 homologs are located anti-sense to a putative toxin, which in turn is never encountered without the small RNA. The secondary structure of PtaRNA1, furthermore, is very similar to that of the FinP anti-sense RNA found on F-like plasmids in Escherichia coli.
A family of non-classical pseudoknots in influenza A and B viruses
A very conserved pseudoknot structure has been shown to fold in influenza virus RNA. The pseudoknot encompasses the 3’ splice site of segment 8 RNA in both influenza A and B viruses. By sequence comparison of influenza virus strains, we derive a consensus motif that defines a novel RNA pseudoknot family. The orientation of the coaxially stacked stems in the influenza pseudoknot differs from that in classical H-pseudoknots. Apart from the size of the loops, the topology of the influenza pseudoknot resembles that of some long-range pseudoknotted conformations. A seed alignment of the influenza pseudoknot family, containing representative strain sequences together with a consensus structure description, has been submitted to the RNA families (Rfam) database.
miRNA, siRNA, piRNA and Argonautes: News in small matters
Since the discovery of the first microRNA (miRNA) family member lin-4 in Caenorhabditis elegans by Lee et al., and RNA interference (RNAi) by Andrew Fire and his colleagues in the 1990s, the new field of regulatory non-coding RNAs has enormously gained momentum and importance. Small regulatory RNAs comprise small interfering RNAs (siRNAs), miRNAs and Piwi-associated small RNAs (piRNAs). Generated from double-stranded RNAs (dsRNAs), siRNAs trigger sequence-specific mRNA decay also known as RNA interference (RNAi). miRNAs in association with Argonaute (AGO) and GW182 proteins, forming the RNA-induced silencing complex (RISC), mediate fine tuning of gene expression and are involved in various biological key processes. An estimate of 500-1000 miRNA genes exist in vertebrates and plants and about 100 in invertebrates. Each miRNA is predicted to target hundreds of mRNAs thus influencing key regulatory mechanisms of the cell. Consequently, deregulated miRNA expression has been suggested to contribute to the initiation and progression of human cancer and other diseases. piRNAs associated with Piwi proteins protect the animal germline from mobile genetic elements, thereby acting as a small RNA based immune system.
Small RNA-mediated regulation at the level of transcript stability
In recent years, bacterial small regulatory RNAs (sRNAs) have been demonstrated to be powerful modulators of gene expression. Whether it is by modulating mRNA functions or protein activities, sRNAs often employ unexpected and extremely diverse mechanisms to modify the genetic output. Although the first sRNAs were characterized as molecules blocking translation of specific target mRNAs, this review will focus on an emerging subset of sRNAs that promote the decay of their target mRNAs. While the outcome resembles the RNAi silencing described in eukaryotes, the mechanism of bacterial sRNAs differs fundamentally. These sRNAs are the subject of intensive studies, which makes them the best characterized sRNAs to date.
P-TEFb stimulates transcription elongation and pre-mRNA splicing through multilateral mechanisms
Promoter-proximal pausing of RNA polymerase II (RNAPII) across the genome has renewed our attention to the early transcriptional events that control the establishment of pausing and the release of RNAPII into a productive transcription elongation. Here, we review our current understanding of the transcriptional cycle by RNAPII with a particular emphasis on the mechanisms that stimulate transcription elongation and cotranscriptional pre-mRNA splicing through an essential transcriptional kinase, the positive transcription elongation factor b (P-TEFb). We illustrate that by targeting a limited set of transcription elongation factors and paused RNAPII molecule during an early phase of transcription, P-TEFb unleashes an extensive crosstalk between transcription apparatus, RNA processing factors and chromatin for optimal production of mRNA.
An unexpected role for RNA in the recognition of DNA by the innate immune system
A central function of our innate immune system is to sense microbial pathogens by the presence of their nucleic acid genomes or their transcriptional or replicative activity. In mammals, a receptor-‐based system is mainly responsible for the detection of these “non-‐ self” nucleic acids. Tremendous progress has been made in the past years to identify host constituents that are required for this intricate task. With regard to the sensing of RNA genome based pathogens by our innate immune system, a picture is emerging that includes certain families of the toll-‐like receptor family (TLR3, TLR7, TLR8) and the RIG-‐ I like helicases (RIG-‐I, MDA5 and LGP2). Genetic loss of function studies implicate that the absence of these pathways can lead to a complete lack of recognition of certain RNA viruses. At the same time, intracellular DNA can also trigger potent innate immune responses, yet the players in this field are less clear. We and another group recently identified a role for RNA polymerase III in the conversion of AT-‐rich DNA into an RNA ligand that is sensed by the RIG-‐I pathway. In this review article, we will discuss the mechanistics and implications of this novel pathway.
Transposon defense in Drosophila somatic cells: A model for distinction of self and non-self in the genome
Genomes need an immune system much like entire organisms, because their integrity is threatened by selfish genetic elements which transpose and proliferate at the cost of the host. Unlike bacteria or viruses, these DNA parasites do not have any particular feature that helps to detect them as foreign sequences within the genome – they have no “antigen” so to speak. Nonetheless, sequence-specific defense mechanisms have evolved: The germ-line piRNAs rely on previous exposure that has left degenerate copies of many transposon-families in certain genomic loci from which small RNA sentinels are produced. In addition, the somatic cells of Drosophila deploy transposon-complementary endo-siRNAs to repress their activity. It was unclear how their precursors are generated or which mechanism leads to preferential targeting of transposons. Several publications now report progress in our understanding of endo-siRNA biogenesis and propose the first models for how “self”-DNA might be distinguished from selfish DNA.
Mimicry of molecular pretenders: The terminal structures of satellites associated with plant RNA viruses
Satellite RNAs (satRNAs) and satellite viruses depend on the replicase complexes provided by their cognate helper viruses and host plants for replication, pretending that they are part of the viral genomes. Although satRNAs and satellite viruses do not share significant nucleotide sequence similarity with the helper viruses, the essential cis-acting elements recognized by the replicase complexes must reside on their genomes, acting as the mimicry for the molecular pretenders. By understanding how this molecular mimicry deceives the helper viruses into supporting the satellites, a significant amount of knowledge of the basic requirements and mechanisms for replication of viruses and satellites has been obtained. Here we review the recent advances in understanding the effects of the cis elements at the termini of satRNAs and satellite viruses on their accumulation. Several well-characterized satellite/helper virus systems, representing the non-coding short satRNAs, mRNA-type long satRNAs, circular satRNAs, and satellite viruses, are compared and contrasted. It is concluded that different satellites may adopt different strategies to exploit the replication/transcription/translation machineries of their helper viruses, and different mimicries may be implemented by the same molecular pretender for different biological functions.
Chloroplast RNA-binding proteins: Repair and regulation of chloroplast transcripts
Chloroplast RNA metabolism has greatly diverged from the cyanobacterial ancestral state. The number of processing sites per transcript has increased and novel processing steps not found in bacteria have been acquired. Whereas many of the processing steps are essential for chloroplast development, it is unclear why such steps evolved at all. In this article, two hypotheses seeking to explain the complexity of chloroplast RNA metabolism are explored: the genomic debugging hypothesis and the nuclear regulation hypothesis. The nuclear-encoded RNA processing factors underlying these two alternative, but not mutually exclusive, hypotheses have very different characteristics. We propose that pentatricopeptide repeat (PPR) proteins, with high sequence specificity and essential roles in various RNA-processing steps, act largely as genomic debuggers. By contrast, the chloroplast ribonucleoproteins (cpRNP), which are strongly modulated by external and internal stimuli, are suggested to be major players in transducing signals to the chloroplast transcript pool.
An exonic splicing enhancer within a bidirectional coding sequence regulates alternative splicing of an antisense mRNA
The discovery of increasing numbers of genes with overlapping sequences highlights the problem of expression in the context of constraining regulatory elements from more than one gene. This study identifies regulatory sequences encompassed within two genes that overlap in an antisense orientation at their 3’ ends. The genes encode the α-thyroid hormone receptor gene (TRα or NR1A1) and Rev-erbα (NR1D1). In mammals TRα pre-mRNAs are alternatively spliced to yield mRNAs encoding functionally antagonistic proteins: TRα1, an authentic thyroid hormone receptor; and TRα2, a non-hormone-binding variant that acts as a repressor. TRα2-specific splicing requires two regulatory elements that overlap with Rev-erbα sequences. Functional mapping of these elements reveals minimal splicing enhancer elements that have evolved within the constraints of the overlapping Rev-erbα sequence. These results provide insight into the evolution of regulatory elements within the context of bidirectional coding sequences. They also demonstrate the ability of the genetic code to accommodate multiple layers of information within a given sequence, an important property of the code recently suggested on theoretical grounds.
Ever since the first genomes of model organisms were sequenced it became obvious that the number of protein coding genes does not reflect organismic complexity. Instead, a relatively constant number of protein coding genes was found in organisms as diverse as C. elegans and humans. While this puzzling observation is still partly unsolved today, research in recent years has shown that RNA-mediated processes may largely solve this dilemma.
Spliceosomal snRNA modifications and their function
Spliceosomal snRNAs are extensively 2'-O-methylated and pseudouridylated. The modified nucleotides are relatively highly conserved across species, and are often clustered in regions of functional importance in pre-mRNA splicing. Over the past decade, the study of the mechanisms and functions of spliceosomal snRNA modifications has intensified. Two independent mechanisms behind these modifications, RNA-independent (protein-only) and RNA-dependent (RNA-guided), have been discovered. The role of spliceosomal snRNA modifications in snRNP biogenesis and spliceosome assembly has also been verified.
Proteome diversification by adenosine to inosine RNA-editing
Nucleotide deamination is a widespread phenomenon frequently leading to a change of the genetic information. Adenosine deaminases that act on RNA (ADARs) convert adenosines to inosines in double-stranded or structured regions of RNAs. Inosines are interpreted as guanosines by most cellular processes and hence, this type of modification can affect the coding potential of an RNA but also its splicing, folding, or stability, to name a few. While originally believed to be a rare event recent bioinformatic screens have demonstrated that RNA-editing by ADARs is widespread and very abundant in mammals. From these screens, editing sites were discovered in both coding and non-coding regions of mRNAs. In this review we focus on RNA-editing events that alter the coding potential of mRNAs and hence lead to the formation of proteins that differ from their genomically encoded versions. We will therefore discuss the role of ADARs in proteome diversification, in particular in the nervous system where editing is most abundant.
In plants, post-transcriptional modification of transcripts includes C-to-U, U-to-C and A-to-I editing. RNA editing in plants is essential, with many mutants impaired in editing of specific sites exhibiting strong deleterious phenotypes, even lethality. The majority of editing in plants occurs in mitochondrial and plastid transcripts, however, A-to-I editing also occurs in cytosolic tRNAs. Here we review recent findings concerning the cellular machineries involved in the different types of editing, recent analysis of the proposed functions for editing, and recent models for its appearance and retention in different plant lineages.
Diverse functions for DNA and RNA editing in the immune system
Polynucleotide DNA and RNA editing enzymes alter nucleic acid sequences and can thereby modify encoded informational content. Two major families of polynucleotide editing enzymes, the AID/APOBEC cytidine deaminases (which catalyze the deamination of cytidine to uridine) and the adenosine deaminases acting on RNA (ADARs, which catalyze the deamination of adenosine to inosine), function in a variety of host defense mechanisms. These enzymes act in innate and adaptive immune pathways, with both host and pathogen targets. DNA editing by the cytidine deaminase AID mediates immunoglobulin somatic hypermutation and class switch recombination, providing the antibody response with the flexibility and diversity to defend against an almost limitless array of varied and rapidly adapting pathogenic challenges. Other cytidine deaminases (APOBEC3) restrict retroviral infection by editing viral retrogenomes. Adenosine deaminases (ADARs) shape innate immune responses by modifying host transcripts that encode immune effectors and their regulators. Here we review current knowledge of polynucleotide DNA and RNA editors with a focus on these and other functions they serve in the immune system.
RNA editing in kinetoplastid protozoa is a post-transcriptional process of uridine insertion or deletion in mitochondrial mRNAs. The process involves two RNA species, the pre-edited mRNA and in most cases a trans-acting guide RNA (gRNA). Sequences within gRNAs define the position and extend of mRNA editing. Both mRNAs and gRNAs are encoded by mitochondrial genes in the kinetoplast DNA (kDNA), which consists of thousands of small circular DNA molecules, called minicircles, encoding thousands of gRNAs, catenated together and with a few mRNA encoding larger circles, the maxicircles, to form a huge DNA network. Editing has been shown to result in translatable mRNAs of bona fide mitochondrial genes as well as novel alternatively edited transcripts that are involved in the maintenance of the kDNA itself. RNA editing occurs within large protein-RNA complexes, editosomes, containing gRNA, preedited and partially edited mRNAs and also structural and catalytically active proteins. Editosomes are diverse in both RNA and protein composition and undergoe structural remodeling during the maturation. The compositional and structural diversity of editosomes further underscores the complexity of the RNA editing process.
RNA nucleotide modifications are typically of low abundance and frequently go unnoticed by standard detection methods of molecular biology and cell biology. With a burst of knowledge intruding from such diverse areas as genomics, structural biology, regulation of gene expression and immunology, it becomes increasingly clear that many exciting functions of nucleotide modifications remain to be explored. It follows in turn that the biology of nucleotide modification and editing is a field poised to rapidly gain importance in a variety of fields. The detection and analysis of nucleotide modifications present a clear limitation in this respect. Here, various methods for detection of nucleotide modifications are discussed based on three discriminating principles, namely physicochemical properties, enzymatic turnover, and chemical reactivity. Because the full extent of nucleotide modification across the various RNA species remains ill understood, emphasis is placed on high-throughput techniques with a potential to screen entire transcriptomes.
Sequence based identification of RNA editing sites
RNA editing diversifies the human transcriptome beyond the genomic repertoire. Recent years have proven a strategy based on genomics and computational sequence analysis as a powerful tool for identification and characterization of RNA editing. In particular, analysis of the human transcriptome has resulted in the identification of thousands of A-to-I editing sites within genomic repeats, as well as a few hundred sites located outside repeats. We review these recent advancements, emphasizing the principles underlying the various methods used. Possible directions for extending these methods are discussed.
A novel tissue-specific alternatively spliced form of the A-to-I RNA editing enzyme ADAR2
ADAR2, a member of the adenosine deaminase family of proteins, is the enzyme that edits the Q/R site in the GluR-B transcript, an important physiological A-to-I editing event. ADAR2 pre-mRNA undergoes a number of known alternative splicing events, affecting its function. Here we describe a novel alternatively spliced exon, located within intron 7 of the human gene, which we term "exon 7a". This alternatively spliced exon is highly conserved in the mammalian ADAR2 gene. It has stop codons in all three frames and is down regulated by NMD. We show that the level of exon 7a inclusion differs between different human tissues, with the highest levels of inclusion in skeletal muscle, heart and testis. In the brain, where the level of editing is known to be high, the level of exon 7a inclusion is low. The new alternative form was also found in supraspliceosomes, which constitute the nuclear pre-mRNA processing machine. The high conservation of the novel ADAR2 alternative exon in mammals indicates a physiological importance for this exon.