Identification of small RNAs abundant in Burkholderia cenocepacia biofilms reveal putative regulators with a potential role in carbon and iron metabolism

Small RNAs play a regulatory role in many central metabolic processes of bacteria, as well as in developmental processes such as biofilm formation. Small RNAs of Burkholderia cenocepacia, an opportunistic pathogenic beta-proteobacterium, are to date not well characterised. To address that, we performed genome-wide transcriptome structure analysis of biofilm grown B. cenocepacia J2315. 41 unannotated short transcripts were identified in intergenic regions of the B. cenocepacia genome. 15 of these short transcripts, highly abundant in biofilms, widely conserved in Burkholderia sp. and without known function, were selected for in-depth analysis. Expression profiling showed that most of these sRNAs are more abundant in biofilms than in planktonic cultures. Many are also highly abundant in cells grown in minimal media, suggesting they are involved in adaptation to nutrient limitation and growth arrest. Their computationally predicted targets include a high proportion of genes involved in carbon metabolism. Expression and target genes of one sRNA suggest a potential role in regulating iron homoeostasis. The strategy used for this study to detect sRNAs expressed in B. cenocepacia biofilms has successfully identified sRNAs with a regulatory function

RNA elements directing in vivo assembly of the 7SK/MePCE/Larp7 transcriptional regulatory snRNP

Through controlling the nuclear level of active positive transcription elongation factor b (P-TEFb), the 7SK small nuclear RNA (snRNA) functions as a key regulator of RNA polymerase II transcription. Together with hexamethylene bisacetamide-inducible proteins 1/2 (HEXIM1/2), the 7SK snRNA sequesters P-TEFb into transcriptionally inactive ribonucleoprotein (RNP). In response to transcriptional stimulation, the 7SK/HEXIM/P-TEFb RNP releases P-TEFb to promote polymerase II-mediated messenger RNA synthesis. Besides transiently associating with HEXIM1/2 and P-TEFb, the 7SK snRNA stably interacts with the La-related protein 7 (Larp7) and the methylphosphate capping enzyme (MePCE). In this study, we used in vivo RNA-protein interaction assays to determine the sequence and structural elements of human 7SK snRNA directing assembly of the 7SK/MePCE/Larp7 core snRNP. MePCE interacts with the short 5'-terminal G1-U4/U106-G111 helix-tail motif and Larp7 binds to the 3'-terminal hairpin and the following U-rich tail of 7SK. The overall RNA structure and some particular nucleotides provide the information for specific binding of MePCE and Larp7. We also demonstrate that binding of Larp7 to 7SK is a prerequisite for in vivo recruitment of P-TEFb, indicating that besides providing stability for 7SK, Larp7 directly participates in P-TEFb regulation. Our results provide further explanation for the frequently observed link between Larp7 mutations and cancer development

Genomic analysis suggests that mRNA destabilization by the microprocessor is specialized for the auto-regulation of Dgcr8.

BackgroundThe Microprocessor, containing the RNA binding protein Dgcr8 and RNase III enzyme Drosha, is responsible for processing primary microRNAs to precursor microRNAs. The Microprocessor regulates its own levels by cleaving hairpins in the 5'UTR and coding region of the Dgcr8 mRNA, thereby destabilizing the mature transcript.Methodology/principal findingsTo determine whether the Microprocessor has a broader role in directly regulating other coding mRNA levels, we integrated results from expression profiling and ultra high-throughput deep sequencing of small RNAs. Expression analysis of mRNAs in wild-type, Dgcr8 knockout, and Dicer knockout mouse embryonic stem (ES) cells uncovered mRNAs that were specifically upregulated in the Dgcr8 null background. A number of these transcripts had evolutionarily conserved predicted hairpin targets for the Microprocessor. However, analysis of deep sequencing data of 18 to 200nt small RNAs in mouse ES, HeLa, and HepG2 indicates that exonic sequence reads that map in a pattern consistent with Microprocessor activity are unique to Dgcr8.Conclusion/significanceWe conclude that the Microprocessor's role in directly destabilizing coding mRNAs is likely specifically targeted to Dgcr8 itself, suggesting a specialized cellular mechanism for gene auto-regulation

Oligonucleotide sequences forming short self-complimentary hairpins can expedite the down-regulation of Coprinopsis cinerea genes

Gene silencing in fungi is often induced by dsRNA hairpin forming constructs the preparation of which can require multiple cloning steps. To simplify gene silencing in the filamentous fungi we have evaluated a high throughput cloning method for target sequences using the homobasidiomycete Coprinopsis cinerea, the GFP reporter and a commercially available vector system. The pSUPER RNAi System™, which was developed for mammalian experiments, exploits the human H1 Polymerase III (Pol III) RNA gene promoter and expedites cloning/expression of specific user-defined oligonucleotide sequences to form short self-complimentary hairpins. Transformation of C. cinerea with pSUPER constructs harboring specific oligonucleotides (19 nt stem length) enabled recovery of transformants with reduced transcripts of the GFP transgene, that were less fluorescent in protein assays and microscopic phenotypes. This technological advance should expedite functional genomic studies in C. cinerea and has wider potential for utility in other homobasidiomycete and filamentous fungi

Short hairpin RNA-mediated knockdown of protein expression in Entamoeba histolytica

<p>Abstract</p> <p>Background</p> <p><it>Entamoeba histolytica </it>is an intestinal protozoan parasite of humans. The genome has been sequenced, but the study of individual gene products has been hampered by the lack of the ability to generate gene knockouts. We chose to test the use of RNA interference to knock down gene expression in <it>Entamoeba histolytica</it>.</p> <p>Results</p> <p>An episomal vector-based system, using the <it>E. histolytica </it>U6 promoter to drive expression of 29-basepair short hairpin RNAs, was developed to target protein-encoding genes in <it>E. histolytica</it>. The short hairpin RNAs successfully knocked down protein levels of all three unrelated genes tested with this system: Igl, the intermediate subunit of the galactose- and N-acetyl-D-galactosamine-inhibitable lectin; the transcription factor URE3-BP; and the membrane binding protein EhC2A. Igl levels were reduced by 72%, URE3-BP by 89%, and EhC2A by 97%.</p> <p>Conclusion</p> <p>Use of the U6 promoter to drive expression of 29-basepair short hairpin RNAs is effective at knocking down protein expression for unrelated genes in <it>Entamoeba histolytica</it>, providing a useful tool for the study of this parasite.</p

RNA interference approaches for treatment of HIV-1 infection.

HIV/AIDS is a chronic and debilitating disease that cannot be cured with current antiretroviral drugs. While combinatorial antiretroviral therapy (cART) can potently suppress HIV-1 replication and delay the onset of AIDS, viral mutagenesis often leads to viral escape from multiple drugs. In addition to the pharmacological agents that comprise cART drug cocktails, new biological therapeutics are reaching the clinic. These include gene-based therapies that utilize RNA interference (RNAi) to silence the expression of viral or host mRNA targets that are required for HIV-1 infection and/or replication. RNAi allows sequence-specific design to compensate for viral mutants and natural variants, thereby drastically expanding the number of therapeutic targets beyond the capabilities of cART. Recent advances in clinical and preclinical studies have demonstrated the promise of RNAi therapeutics, reinforcing the concept that RNAi-based agents might offer a safe, effective, and more durable approach for the treatment of HIV/AIDS. Nevertheless, there are challenges that must be overcome in order for RNAi therapeutics to reach their clinical potential. These include the refinement of strategies for delivery and to reduce the risk of mutational escape. In this review, we provide an overview of RNAi-based therapies for HIV-1, examine a variety of combinatorial RNAi strategies, and discuss approaches for ex vivo delivery and in vivo delivery

Techniques for RNA in vivo imaging in plants

Since the discovery of small RNAs and RNA silencing, RNA biology has taken a centre stage in cell and developmental biology. Small RNAs, but also mRNAs and other types of cellular and viral RNAs are processed at specific subcellular localizations. To fully understand cellular RNA metabolism and the various processes influenced byit, techniques are required that permit the sequence-specific tracking of RNAs in living cells. A variety of methods for RNA visualization have been developed since the 1990s, but plant cells pose particular challenges and not all approaches are applicable to them. On the other hand, plant RNA metabolism is particularly diverse and RNAs are even transported between cells, so RNA imaging can potentially provide many valuable insights into plant function at the cellular and tissue level. This Short Review briefly introduces the currently available techniques for plant RNA in vivo imaging and discusses their suitability for different biological questions.PostprintPeer reviewe

Trans-inhibition of HIV-1 by a long hairpin RNA expressed within the viral genome

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) can be inhibited by means of RNA silencing or interference (RNAi) using synthetic short interfering RNAs (siRNAs) or gene constructs encoding short hairpin RNAs (shRNAs) or long hairpin RNAs (lhRNAs). The use of siRNA and shRNA as antiviral therapeutic is limited because of the emergence of viral escape mutants. This problem is theoretically prevented by intracellular expression of lhRNAs generating multiple siRNAs that target the virus simultaneously, thus reducing the chance of viral escape. However, gene constructs encoding lhRNA molecules face problems with delivery to the right cells in an infected individual. In order to solve this problem, we constructed an HIV-1 variant with a 300 bp long hairpin structure in the 3' part of the genome corresponding to the Nef gene (HIV-lhNef). RESULTS: Intriguingly, HIV-lhNef potently inhibited wild-type HIV-1 production in trans. However, HIV-lhNef demonstrated a severe production and replication defect, which we were able to solve by selecting spontaneous virus variants with truncated hairpin structures. Although these escape variants lost the ability to trans-inhibit HIV-1, they effectively outgrew the wild-type virus in competition experiments in SupT1 cells. CONCLUSION: Expression of the lhNef hairpin within the HIV-1 genome results in potent trans-inhibition of wild-type HIV-1. Although the mechanism of trans-inhibition is currently unknown, it remains of interest to study the molecular details because the observed effect is extremely potent. This may have implications for the development of virus strains to be used as live-attenuated virus vaccines