Identification of small non-coding RNAs from mitochondria and chloroplasts

Small non-protein-coding RNAs (ncRNAs) have been identified in a wide spectrum of organisms ranging from bacteria to humans. In eukarya, systematic searches for ncRNAs have so far been restricted to the nuclear or cytosolic compartments of cells. Whether or not small stable non-coding RNA species also exist in cell organelles, in addition to tRNAs or ribosomal RNAs, is unknown. We have thus generated cDNA libraries from size-selected mammalian mitochondrial RNA and plant chloroplast RNA and searched for small ncRNA species in these two types of DNA-containing cell organelles. In total, we have identified 18 novel candidates for organellar ncRNAs in these two cellular compartments and confirmed expression of six of them by northern blot analysis or RNase A protection assays. Most candidate ncRNA genes map to intergenic regions of the organellar genomes. As found previously in bacteria, the presumptive ancestors of present-day chloroplasts and mitochondria, we also observed examples of antisense ncRNAs that potentially could target organelle-encoded mRNAs. The structural features of the identified ncRNAs as well as their possible cellular functions are discussed. The absence from our libraries of abundant small RNA species that are not encoded by the organellar genomes suggests that the import of RNAs into cell organelles is of very limited significance or does not occur at all.     

综述: 新型长链非编码RNA(lncRNA)的生物信息学研究进展

非编码RNA (noncoding RNA,ncRNA)是指不被翻译成蛋白质的一类RNA,近几年来关于它们的功能研究越来越引起人们的重视．现在已经发现了一些中小型ncRNA,比如microRNA、snoRNA、tRNA等,但是关于长ncRNA(lncRNA)的研究还不够完善．本篇综述回顾了 ncRNA特别是 lncRNA的生物信息学研究进展,包括它们的研究历程、基本特点、与疾病的关系,以及对已有的预测非编码RNA的计算机方法进行了分析和比较,并且介绍了利用机器学习模型整合新一代高通量测序数据的方法．     

A simple method of examining retroviral species produced by infected cells

A simple and rapid method to examine the retroviral species present in the culture supernatant of productively infected cells is described. The method involves purification of viral genomic RNA directly from the culture supernatant and examination of that RNA by Northern or dot blot analysis. The method provides qualitative and quantitative information about the RNA species present and is particularly valuable for the detection of genetic variants in the population.     

Decoding microRNA drivers in atherosclerosis

An estimated 97% of the human genome consists of non-protein-coding sequences. As our understanding of genome regulation improves, this has led to the characterization of a diverse array of non-coding RNAs (ncRNA). Among these, micro-RNAs (miRNAs) belong to the short ncRNA class (22–25 nucleotides in length), with approximately 2500 miRNA genes encoded within the human genome. From a therapeutic perspective, there is interest in exploiting miRNA as biomarkers of disease progression and response to treatments, as well as miRNA mimics/repressors as novel medicines. miRNA have emerged as an important class of RNA master regulators with important roles identified in the pathogenesis of atherosclerotic cardiovascular disease. Atherosclerosis is characterized by a chronic inflammatory build-up, driven largely by low-density lipoprotein cholesterol accumulation within the artery wall and vascular injury, including endothelial dysfunction, leukocyte recruitment and vascular remodelling. Conventional therapy focuses on lifestyle interventions, blood pressure-lowering medications, high-intensity statin therapy and antiplatelet agents. However, a significant proportion of patients remain at increased risk of cardiovascular disease. This continued cardiovascular risk is referred to as residual risk. Hence, a new drug class targeting atherosclerosis could synergise with existing therapies to optimise outcomes. Here, we review our current understanding of the role of ncRNA, with a focus on miRNA, in the development and progression of atherosclerosis, highlighting novel biological mechanisms and therapeutic avenues.     

Detection of Leishmania RNA virus 1 proteins.

Polyclonal antiserum was raised against the peak viral fraction of a sucrose gradient from LRV1-4-infected cells and used in Western immunoblot analysis to identify viral proteins from various isolates. Consistent with this result, in vitro-translated protein from cloned RNA was immunoprecipitated with the same antiserum. The putative capsid at times appeared as a doublet; relative amounts of the two species varied, depending on the method of purification.     

Protocol for nearly full-length sequencing of HIV-1 RNA from plasma

Nearly full-length genome sequencing of HIV-1 using peripheral blood mononuclear cells (PBMC) DNA as a template for PCR is now a relatively routine laboratory procedure. However, this has not been the case when using virion RNA as the template and this has made full genome analysis of circulating viruses difficult. Therefore, a well-developed procedure for sequencing of full-length HIV-1 RNA directly from plasma was needed. Plasma from U.S. donors representing a range of viral loads (VL) was used to develop the assay. RNA was extracted from plasma and reverse-transcribed. Two or three overlapping regions were PCR amplified to cover the entire viral genome and sequenced for verification. The success of the procedure was sensitive to VL but was routinely successful for VL greater than 10(5) and the rate declined in proportion to the VL. While the two-amplicon strategy had an advantage of increasing the possibility of amplifying a single species of HIV-1, the three-amplicon strategy was more successful in amplifying samples with low viral loads. This protocol provides a useful tool for molecular analysis to understand the HIV epidemic and pathogenesis, as well as diagnosis, therapy and future vaccine strategies.     

Methodological obstacles in knocking down small noncoding RNAs

In the recent past, several thousand noncoding RNA (ncRNA) genes have been predicted within eukaryal genomes. However, for their functional analysis only a few high-throughput methods are currently available to knock down selected ncRNA species, such as microRNAs, which are targeted by antisense probes, termed antagomirs. We thus compared the efficiencies of four knockdown strategies, previously mainly employed for the analysis of protein-coding genes, to study the function of ncRNAs, in particular, small nucleolar RNAs (snoRNAs). Thereby, the class of snoRNAs represents one of the most abundant ncRNA species. The majority of snoRNAs has been shown to mediate nucleotide modifications by targeting ribosomal RNAs (rRNAs) through complementary antisense elements. However, some snoRNAs, termed “orphan snoRNAs,” lack telltale complementarities to rRNAs and thus their function remains elusive. We therefore applied RNA interference (RNAi), locked nucleic acid (LNA), or peptide nucleic acid antisense approaches, as well as a ribozyme-based strategy to knock down a snoRNA. As a proof of principle, we targeted the canonical U81 snoRNA, which has been shown to mediate modification of nucleotide A₃₉₁ within eukaryal 28S rRNA. Our results demonstrate that while RNAi is an unsuitable tool for snoRNA knockdown, a ribozyme-based strategy, as well as an LNA-antisense oligonucleotide approach, resulted in a decrease of U81 snoRNA expression levels up to 60%. However, no concomitant decrease in enzymatic activity of U81 snoRNA was observed, indicating that improvement of more efficient knockdown techniques for ncRNAs will be required in the future.     

Isolation of viral ribonucleoprotein complexes from infected cells by tandem affinity purification

The biochemical purification and analysis of viral ribonucleoprotein complexes (RNPs) of negative-strand RNA viruses is hampered by the lack of suitable tags that facilitate specific enrichment of these complexes. We therefore tested whether fusion of the tandem-affinity-purification (TAP) tag to the main component of viral RNPs, the nucleoprotein, might allow the isolation of these RNPs from cells. We constitutively expressed TAP-tagged nucleoprotein of Borna disease virus (BDV) in cells persistently infected with this virus. The TAP-tagged bait was efficiently incorporated into viral RNPs, did not interfere with BDV replication and was also packaged into viral particles. Native purification of the tagged protein complexes from BDV-infected cells by two consecutive affinity columns resulted in the isolation of several viral proteins, which were identified by MS analysis as the matrix protein, the two forms of the nucleoprotein and the phosphoprotein. In addition to the viral proteins, RT-PCR analysis revealed the presence of viral genomic RNA. Introduction of further protease cleavage sites within the TAP-tag significantly increased the purification yield. These results demonstrate that purification of TAP-tagged viral RNPs is possible and efficient, and may therefore provide new avenues for biochemical and functional studies of these complexes.     

Isolation of specific RNA-binding proteins using the streptomycin-binding RNA aptamer

Here we report a simple and cheap one-step affinity purification protocol for isolating RNAs or proteins that interact with selected functional RNAs. The streptomycin-binding aptamer, termed 'StreptoTag,' is embedded in or fused to either end of any RNA of interest. The resulting hybrid RNA can then be immobilized on a streptomycin affinity matrix. When a complex protein mixture or total cellular lysate is applied to the matrix, subsequent elution with free streptomycin allows efficient recovery of specific ribonucleoprotein or RNA-RNA complexes. The method was successfully used to purify yeast and phage RNA-binding proteins and group II intron, viral and bacterial noncoding RNA (ncRNA)-binding proteins. The selective enrichment of bacterial mRNAs that bind ncRNAs has also been demonstrated. Once the affinity matrix, the RNA construct and the protein extracts have been prepared, the experimental procedure can be performed in 1-2 h.     

Plant noncoding RNA gene discovery by "single-genome comparative genomics"

Plant genomes have undergone multiple rounds of duplications that contributed massively to the growth of gene families. The structure of resulting families has been studied in depth for protein-coding genes. However, little is known about the impact of duplications on noncoding RNA (ncRNA) genes. Here we perform a systematic analysis of duplicated regions in the rice genome in search of such ncRNA repeats. We observe that, just like their protein counterparts, most ncRNA genes have undergone multiple duplications that left visible sequence conservation footprints. The extent of ncRNA gene duplication in plants is such that these sequence footprints can be exploited for the discovery of novel ncRNA gene families on a large scale. We developed an SVM model that is able to retrieve likely ncRNA candidates among the 100,000+ repeat families in the rice genome, with a reasonably low false-positive discovery rate. Among the nearly 4000 ncRNA families predicted by this means, only 90 correspond to putative snoRNA or miRNA families. About half of the remaining families are classified as structured RNAs. New candidate ncRNAs are particularly enriched in UTR and intronic regions. Interestingly, 89% of the putative ncRNA families do not produce a detectable signal when their sequences are compared to another grass genome such as maize. Our results show that a large fraction of rice ncRNA genes are present in multiple copies and are species-specific or of recent origin. Intragenome comparison is a unique and potent source for the computational annotation of this major class of ncRNA.