Exogenous and endogenous dsRNAs perceived by plant Dicer-like 4 protein in the RNAi-depleted cellular context.

BACKGROUND: In plants, RNase III Dicer-like proteins (DCLs) act as sensors of dsRNAs and process them into short 21- to 24-nucleotide (nt) (s)RNAs. Plant DCL4 is involved in the biogenesis of either functional endogenous or exogenous (i.e. viral) short interfering (si)RNAs, thus playing crucial antiviral roles. METHODS: In this study we expressed plant DCL4 in Saccharomyces cerevisiae, an RNAi-depleted organism, in which we could highlight the role of dicing as neither Argonautes nor RNA-dependent RNA polymerase is present. We have therefore tested the DCL4 functionality in processing exogenous dsRNA-like substrates, such as a replicase-assisted viral replicon defective-interfering RNA and RNA hairpin substrates, or endogenous antisense transcripts. RESULTS: DCL4 was shown to be functional in processing dsRNA-like molecules in vitro and in vivo into 21- and 22-nt sRNAs. Conversely, DCL4 did not efficiently process a replicase-assisted viral replicon in vivo, providing evidence that viral RNAs are not accessible to DCL4 in membranes associated in active replication. Worthy of note, in yeast cells expressing DCL4, 21- and 22-nt sRNAs are associated with endogenous loci. CONCLUSIONS: We provide new keys to interpret what was studied so far on antiviral DCL4 in the host system. The results all together confirm the role of sense/antisense RNA-based regulation of gene expression, expanding the sense/antisense atlas of S. cerevisiae. The results described herein show that S. cerevisiae can provide insights into the functionality of plant dicers and extend the S. cerevisiae tool to new biotechnological applications.

One-Pot Production of RNA in High Yield and Purity Through Cleaving Tandem Transcripts.

There is an increasing demand for efficient and robust production of short RNA molecules in both pharmaceutics and research. A standard method is in vitro transcription by T7 RNA polymerase. This method is sequence-dependent on efficiency and is limited to products longer than ~12 nucleotides. Additionally, the native initiation sequence is required to achieve high yields, putting a strain on sequence variability. Deviations from this sequence can lead to side products, requiring laborious purification, further decreasing yield. We here present transcribing tandem repeats of the target RNA sequence followed by site-specific cleavage to obtain RNA in high purity and yield. This approach makes use of a plasmid DNA template and RNase H-directed cleavage of the transcript. The method is simpler and faster than previous protocols, as it can be performed as one pot synthesis and provides at the same time higher yields of RNA.

Comparative Evaluation of Lipofectamine and Dendrimer for Transfection of Short RNA Into Human T47D and MCF-10A Cell Lines.

Purpose: Non-viral transfection approaches are extensively used in cancer therapy. The future of cancer therapy lies on targeted and efficient drug/gene delivery. The aim of this study was to determine the transfection yields of two commercially available transfection reagents (i.e. Lipofectamine 2000, as a cationic lipid and PAMAM G5, as a cationic dendrimer) in two breast cell lines: cancerous cells (T47D) and non-cancerous ones (MCF-10A). Methods: We investigated the efficiencies of Lipofectamine 2000 and PAMAM G5 for transfection/delivery of a labeled short RNA into T47D and MCF-10A. In addition to microscopic assessments, the cellular uptakes of the complexes (fluorescein tagged-scrambled RNA with Lipofectamine or PAMAM dendrimer) were quantified by flow cytometry. Furthermore, the safety of the mentioned reagents was assessed by measuring cell necrosis through the cellular PI uptake. Results: Our results showed significantly better efficiencies of Lipofectamine compared to PAMAM dendrimer for short RNA transfection in both cell types. On the other hand, MCF-10A resisted more than T47D to the toxicity of higher concentrations of the transfection reagents. Conclusion: Altogether, our research demonstrated a route for comprehensive epigenetic modification of cancer cells and depicted an approach to efficient drug delivery, which eventually improves both short RNA-based biopharmaceutical industry and non-viral strategies in epigenetic therapy.

Genome-wide identification and characterization of transfer RNA-derived small RNAs in Plasmodium falciparum.

BACKGROUND: Transfer RNA (tRNA)-derived fragments (tRFs) have been widely identified in nature, functioning in diverse biological and pathological situations. Yet, the presence of these small RNAs in Plasmodium spp. remains unknown. Systematic identification and characterization of tRFs is therefore highly needed to understand further their roles in Plasmodium parasites, particularly in the virulent Plasmodium falciparum parasite. RESULTS: Genome-wide small RNAs with sizes ranging from 18-30 nucleotides from P. falciparum were deep-sequenced via Illumina HiSeq 2000 technology. In-depth analysis revealed the presence of a vast number of small RNAs originating from tRNA-coding genes, responsible for 22.4% of the total reads as the second predominant group. Three P. falciparum-derived tRF types (ptRFs) were identified as 5'ptRFs, mid-ptRFs and 3'ptRFs. The majority (90%) of ptRFs were derived from tRNAs that coded eight amino acids: Pro, Phe, Asn, Gly, Cys, Gln, His and Ala. Stem-loop reverse transcription polymerase chain reaction further confirmed the presence of tRFs in the blood stages of P. falciparum. Four new motifs with an enriched G/C feature were determined at cleavage sites that might guide the generation of ptRFs. CONCLUSIONS: To our knowledge, this is the first report of a genome-wide investigation of ptRFs from Plasmodium species. The identification of ptRFs reveals a complex small RNA system manipulated by the malaria parasite, and might promote research on the function of tRFs in the pathogenesis of Plasmodium infections.

CRISPR/Cas9-mediated MSTN gene editing induced mitochondrial alterations in C2C12 myoblast cells

Background: Myostatin (MSTN) negatively regulates muscle mass and is a potent regulator of energy metabolism. However, MSTN knockout have affect mitochondrial function. This research assessed the mitochondrial energy metabolism of Mstn−/+ KO cells, and wondered whether the mitochondria biogenesis are affected. Results: In this study, we successfully achieved Mstn knockout in skeletal muscle C2C12 cells using a CRISPR/Cas9 system and measured proliferation and differentiation using the Cell-Counting Kit-8 assay and qPCR, respectively. We found that MSTN dysfunction could promote proliferation and differentiation compared with the behaviour of wild-type cells. Moreover, Mstn KO induced an increase in KIF5B expression. The mitochondrial content was significantly increased in Mstn KO C2C12 cells, apparently associated with the increases in PGC-1α, Cox1, Cox2, ND1 and ND2 expression. However, no differences were observed in glucose consumption and lactate production. Interestingly, Mstn KO C2C12 cells showed an increase in IL6 and a decrease in TNF-1α levels. Conclusion: These findings indicate that MSTN regulates mitochondrial biogenesis and metabolism. This gene-editing cells provided favourable evidence for animal breeding and metabolic diseases.