A cap 0-dependent mRNA capture method to analyze the yeast transcriptome.

Analysis of the protein coding transcriptome by the RNA sequencing requires either enrichment of the desired fraction of coding transcripts or depletion of the abundant non-coding fraction consisting mainly of rRNA. We propose an alternative mRNA enrichment strategy based on the RNA-binding properties of the human IFIT1, an antiviral protein recognizing cap 0 RNA. Here, we compare for Saccharomyces cerevisiae an IFIT1-based mRNA pull-down with yeast targeted rRNA depletion by the RiboMinus method. IFIT1-based RNA capture depletes rRNA more effectively, producing high quality RNA-seq data with an excellent coverage of the protein coding transcriptome, while depleting cap-less transcripts such as mitochondrial or some non-coding RNAs. We propose IFIT1 as a cost effective and versatile tool to prepare mRNA libraries for a variety of organisms with cap 0 mRNA ends, including diverse plants, fungi and eukaryotic microbes.

Sequence-specific endoribonucleases.

Ribonucleases are nucleolytic enzymes that commonly occur in living organisms and act by cleaving RNA molecules. These enzymes are involved in basic cellular processes, including the RNA maturation that accompanies the formation of functional RNAs, as well as RNA degradation that enables removal of defective or dangerous molecules or ones that have already fulfilled their cellular functions. RNA degradation is also one of the main processes that determine the amount of transcripts in the cell and thus it makes an important element of the gene expression regulation system. Ribonucleases can catalyse reactions involving RNA molecules containing specific sequences, structures or sequences within a specific structure, they can also cut RNAs non-specifically. In this article, we discuss ribonucleases cleaving the phosphodiester bond inside RNA molecules within or close to particular sequences. We also present examples of protein engineering of ribonucleases towards the development of molecular tools for sequence-specific cleavage of RNA.

RNApathwaysDB--a database of RNA maturation and decay pathways.

Many RNA molecules undergo complex maturation, involving e.g. excision from primary transcripts, removal of introns, post-transcriptional modification and polyadenylation. The level of mature, functional RNAs in the cell is controlled not only by the synthesis and maturation but also by degradation, which proceeds via many different routes. The systematization of data about RNA metabolic pathways and enzymes taking part in RNA maturation and degradation is essential for the full understanding of these processes. RNApathwaysDB, available online at http://iimcb.genesilico.pl/rnapathwaysdb, is an online resource about maturation and decay pathways involving RNA as the substrate. The current release presents information about reactions and enzymes that take part in the maturation and degradation of tRNA, rRNA and mRNA, and describes pathways in three model organisms: Escherichia coli, Saccharomyces cerevisiae and Homo sapiens. RNApathwaysDB can be queried with keywords, and sequences of protein enzymes involved in RNA processing can be searched with BLAST. Options for data presentation include pathway graphs and tables with enzymes and literature data. Structures of macromolecular complexes involving RNA and proteins that act on it are presented as 'potato models' using DrawBioPath-a new javascript tool.

Identification of stable, high copy number, medium-sized RNA degradation intermediates that accumulate in plants under non-stress conditions.

It is becoming increasingly evident that the RNA degradome is a crucial component of the total cellular RNA pool. Here, we present an analysis of the medium-sized RNAs (midi RNAs) that form in Arabidopsis thaliana. Our analyses revealed that the midi RNA fraction contained mostly 20-70-nt-long fragments derived from various RNA species, including tRNA, rRNA, mRNA and snRNA. The majority of these fragments could be classified as stable RNA degradation intermediates (RNA degradants). Using two dimensional polyacrylamide gel electrophoresis, we demonstrated that high copy number RNA (hcn RNA) degradants appear in plant cells not only during stress, as it was earlier suggested. They are continuously produced also under physiological conditions. The data collected indicated that the accumulation pattern of the hcn RNA degradants is organ-specific and can be affected by various endogenous and exogenous factors. In addition, we demonstrated that selected degradants efficiently inhibit translation in vitro. Thus, the results of our studies suggest that hcn RNA degradants are likely to be involved in the regulation of gene expression in plants.

RNA degradome--its biogenesis and functions.

RNA degradation is among the most fundamental processes that occur in living cells. The continuous decay of RNA molecules is associated not only with nucleotide turnover, but also with transcript maturation and quality control. The efficiency of RNA decay is ensured by a broad spectrum of both specific and non-specific ribonucleases. Some of these ribonucleases participate mainly in processing primary transcripts and in RNA quality control. Others preferentially digest mature, functional RNAs to yield a variety of molecules that together constitute the RNA degradome. Recently, it has become increasingly clear that the composition of the cellular RNA degradome can be modulated by numerous endogenous and exogenous factors (e.g. by stress). In addition, instead of being hydrolyzed to single nucleotides, some intermediates of RNA degradation can accumulate and function as signalling molecules or participate in mechanisms that control gene expression. Thus, RNA degradation appears to be not only a process that contributes to the maintenance of cellular homeostasis but also an underestimated source of regulatory molecules.

2D-PAGE as an effective method of RNA degradome analysis.

The continuously growing interest in small regulatory RNA exploration is one of the important factors that have inspired the recent development of new high throughput techniques such as DNA microarrays or next generation sequencing. Each of these methods offers some significant advantages but at the same time each of them is expensive, laborious and challenging especially in terms of data analysis. Therefore, there is still a need to develop new analytical methods enabling the fast, simple and cost-effective examination of the complex RNA mixtures. Recently, increasing attention has been focused on the RNA degradome as a potential source of riboregulators. Accordingly, we attempted to employ a two-dimensional gel electrophoresis as a quick and uncomplicated method of profiling RNA degradome in plant or human cells. This technique has been successfully used in proteome analysis. However, its application in nucleic acids studies has been very limited. Here we demonstrate that two dimensional electrophoresis is a technique which allows one to quickly and cost-effectively identify and compare the profiles of 10-90 nucleotide long RNA accumulation in various cells and organs.

Specific interaction of zinc finger protein Com with RNA and the crystal structure of a self-complementary RNA duplex recognized by Com.

The bacteriophage Mu Com is a small zinc finger protein that binds to its cognate mom mRNA and activates its translation. The Mom protein, in turn, elicits a chemical modification (momification) of the bacteriophage genome, rendering the DNA resistant to cleavage by bacterial restriction endonucleases, and thereby protecting it from defense mechanisms of the host. We examined the basis of specificity in Com-RNA interactions by in vitro selection and probing of RNA structure. We demonstrated that Com recognizes a sequence motif within a hairpin-loop structure of its target RNA. Our data support the model of Com interaction with mom mRNA, in which Com binds to the short hairpin structure proximal to the so-called translation inhibition structure. We also observed that Com binds its target motif weakly if it is within an RNA duplex. These results suggest that the RNA structure, in addition to its sequence, is crucial for Com to recognize its target and that RNA conformational changes may constitute another level of Mom regulation. We determined a crystal structure of a Com binding site variant designed to form an RNA duplex preferentially. Our crystal model forms a 19-mer self-complementary double helix composed of the canonical and non-canonical base pairs. The helical parameters of crystalized RNA indicate why Com may bind it more weakly than a monomeric hairpin form.

RRMdb-an evolutionary-oriented database of RNA recognition motif sequences.

RNA-recognition motif (RRM) is an RNA-interacting protein domain that plays an important role in the processes of RNA metabolism such as the splicing, editing, export, degradation, and regulation of translation. Here, we present the RNA-recognition motif database (RRMdb), which affords rapid identification and annotation of RRM domains in a given protein sequence. The RRMdb database is compiled from ~57 000 collected representative RRM domain sequences, classified into 415 families. Whenever possible, the families are associated with the available literature and structural data. Moreover, the RRM families are organized into a network of sequence similarities that allows for the assessment of the evolutionary relationships between them.

Crystal structure of human Acinus RNA recognition motif domain.

Acinus is an abundant nuclear protein involved in apoptosis and splicing. It has been implicated in inducing apoptotic chromatin condensation and DNA fragmentation during programmed cell death. Acinus undergoes activation by proteolytic cleavage that produces a truncated p17 form that comprises only the RNA recognition motif (RRM) domain. We have determined the crystal structure of the human Acinus RRM domain (AcRRM) at 1.65 Å resolution. It shows a classical four-stranded antiparallel ß-sheet fold with two flanking α-helices and an additional, non-classical α-helix at the C-terminus, which harbors the caspase-3 target sequence that is cleaved during Acinus activation. In the structure, the C-terminal α-helix partially occludes the potential ligand binding surface of the ß-sheet and hypothetically shields it from non-sequence specific interactions with RNA. Based on the comparison with other RRM-RNA complex structures, it is likely that the C-terminal α-helix changes its conformation with respect to the RRM core in order to enable RNA binding by Acinus.

RNA partial degradation problem: motivation, complexity, algorithm.

Studies conducted during the last decade unexpectedly revealed several new biological functions of RNA molecules. The involvement of RNA in many complex processes requires highly effective systems controlling its accumulation. In this context, the mechanisms of degradation appear as one of the most important factors influencing RNA activity. Here, we present our first attempt to describe the RNA degradation process using bioinformatics methods. Based on the obtained data, we propose a formulation of a new problem, called RNA Partial Degradation Problem (RNA PDP) and the algorithm that is capable of reconstructing an RNA molecule using the results of biochemical analysis of its degradation. In addition, we present the results of biochemical experiments and computational tests.