siRNAs targeting mouse myostatin

Eight different mouse myostatin small interfering RNA (siRNAs) were synthesized and tested. Five siRNAs showed a pronounced biological effect reducing myostatin mRNA content. For two of them, the myostatin mRNA level was reduced 3- and 4-fold, respectively. The obtained siRNAs can be used for study of biological effects of myostatin, both in vitro and in vivo.     

Myostatin short interfering hairpin RNA gene transfer increases skeletal muscle mass

BACKGROUND: Myostatin negatively regulates skeletal muscle growth. Myostatin knockout mice exhibit muscle hypertrophy and decreased interstitial fibrosis. We investigated whether a plasmid expressing a short hairpin interfering RNA (shRNA) against myostatin and transduced using electroporation would increase local skeletal muscle mass. METHODS: Short interfering RNAs (siRNAs) targeting myostatin were co-transfected with a myostatin-expressing plasmid into HEK293 cells and identified for myostatin silencing by Western blot. Corresponding shRNAs were cloned into plasmid shRNA expression vectors. Myostatin or a randomer negative control shRNA plasmid was injected and electroporated into the tibialis anterior or its contralateral muscle, respectively, of nine rats that were sacrificed after 2 weeks. Six other rats received a beta-galactosidase reporter plasmid and were sacrificed at 1, 2, and 4 weeks. Uptake of plasmid was examined by beta-galactosidase expression, whereas myostatin expression was determined by real-time polymerase chain reaction (PCR) and Western blotting. Muscle fiber size was determined by histochemistry. Satellite cell proliferation was determined by PAX7 immunohistochemistry. Myosin heavy chain type II (MHCII) expression was determined by Western blot. RESULTS: beta-Galactosidase reporter plasmid was expressed at 1 and 2 weeks but diminished by 4 weeks in tibialis anterior skeletal muscle. Myostatin shRNA reduced myostatin mRNA and protein expression by 27 and 48%, respectively. Tibialis anterior weight, fiber size, and MHCII increased by 10, 34, and 38%, respectively. Satellite cell number was increased by over 2-fold. CONCLUSIONS: This is the first demonstration that myostatin shRNA gene transfer is a potential strategy to increase muscle mass.     

Gene regulation by non-coding RNAs

Abstract

The past two decades have seen an explosion in research on non-coding RNAs and their physiological and pathological functions. Several classes of small (20–30 nucleotides) and long (>200 nucleotides) non-coding RNAs have been firmly established as key regulators of gene expression in myriad processes ranging from embryonic development to innate immunity. In this review, we focus on our current understanding of the molecular mechanisms underlying the biogenesis and function of small interfering RNAs (siRNAs), microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs). In addition, we briefly review the relevance of small and long non-coding RNAs to human physiology and pathology and their potential to be exploited as therapeutic agents.     

Myostatin gene silencing by RNA interference in chicken embryo fibroblast cells

Myostatin (MSTN), a member of transforming growth factor-β (TGF-β) superfamily, is a negative regulator of the skeletal muscle growth, and suppresses the proliferation and differentiation of myoblast cells. Dysfunction of MSTN gene either by natural mutation or genetic manipulation (knockout or knockdown) has been reported to interrupt its proper function and to increase the muscle mass in many mammalian species. RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful tool for gene knockdown studies. In the present study transient silencing of MSTN gene in chicken embryo fibroblast cells was evaluated using five different shRNA expression constructs. We report here up to 68% silencing of myostatin mRNA using these shRNA constructs in transiently transfected fibroblasts (p<0.05). This was, however, associated with induction of interferon responsive genes (OAS1, IFN-β) (3.7-64 folds; p<0.05). Further work on stable expression of antimyostatin shRNA with minimum interferon induction will be of immense value to increase the muscle mass in the transgenic animals.     

microPrimer: the biogenesis and function of microRNA

Discovered in nematodes in 1993, microRNAs (miRNAs) are non-coding RNAs that are related to small interfering RNAs (siRNAs), the small RNAs that guide RNA interference (RNAi). miRNAs sculpt gene expression profiles during plant and animal development. In fact, miRNAs may regulate as many as one-third of human genes. miRNAs are found only in plants and animals, and in the viruses that infect them. miRNAs function very much like siRNAs, but these two types of small RNAs can be distinguished by their distinct pathways for maturation and by the logic by which they regulate gene expression.     

Dicer-dependent biogenesis of small RNAs derived from 7SL RNA

It has been reported that decreased Dicer expression leads to Alu RNAs accumulation in human retinal pigmented epithelium cells, and Dicer may process the endogenous SINE/B1 RNAs (the rodent equivalent of the primate Alu RNAs) into small interfering RNAs (siRNAs). In this study, we aimed to address whether Dicer can process Alu RNAs and their common ancestor, 7SL RNA. Using Solexa sequencing technology, we showed that Alu-derived small RNAs accounted for 0.6% of the total cellular small RNAs in HepG2.2.15 cells, and the abundance decreased when Dicer was knocked down. However, Alu-derived small RNAs showed different characteristics from miRNAs and siRNAs, the classic Dicer-processed products. Interestingly, we found that small RNAs derived from 7SL RNA accounted for 3.1% of the total cellular small RNAs in the control cells, and the abundance dropped about 3.4 folds in Dicer knockdown cells. Dicer-dependent biogenesis of 7SL RNA-derived small RNAs was validated by northern blotting. In vitro cleavage assay using the recombinant human Dicer protein also showed that synthetic 7SL RNA was processed by Dicer into fragments of different lengths. Further functional analysis suggested that 7SL RNA-derived small RNAs do not function like miRNAs, neither do they regulate the expression of 7SL RNA. In conclusion, the current study demonstrated that Dicer can process 7SL RNA, however, the biological significance remains to be elucidated.     

Inhibition of HIV-1 multiplication by antisense U7 snRNAs and siRNAs targeting cyclophilin A

Human immunodeficiency virus 1 (HIV-1) multiplication depends on a cellular protein, cyclophilin A (CyPA), that gets integrated into viral particles. Because CyPA is not required for cell viability, we attempted to block its synthesis in order to inhibit HIV-1 replication. For this purpose, we used antisense U7 small nuclear RNAs (snRNAs) that disturb CyPA pre-mRNA splicing and short interfering RNAs (siRNAs) that target CyPA mRNA for degradation. With dual-specificity U7 snRNAs targeting the 3′ and 5′ splice sites of CyPA exons 3 or 4, we obtained an efficient skipping of these exons and a strong reduction of CyPA protein. Furthermore, short interfering RNAs targeting two segments of the CyPA coding region strongly reduced CyPA mRNA and protein levels. Upon lentiviral vector-mediated transduction, prolonged antisense effects were obtained for both types of antisense RNAs in the human T-cell line CEM-SS. These transduced CEM-SS cells showed a delayed, and for the siRNAs also reduced, HIV-1 multiplication. Since the two types of antisense RNAs function by different mechanisms, combining the two approaches may result in a synergistic effect.     

SRNAome and degradome sequencing analysis reveals specific regulation of sRNA in response to chilling injury in tomato fruit

Plant genomes encode diverse small RNA classes that function in distinct gene‐silencing pathways. To elucidate the intricate regulation of microRNAs (miRNAs) and endogenous small‐interfering RNAs (siRNAs) in response to chilling injury in tomato fruit, the deep sequencing and bioinformatic methods were combined to decipher the small RNAs landscape in the control and chilling‐injured groups. Except for the known miRNAs and ta‐siRNAs, 85 novel miRNAs and 5 ta‐siRNAs members belonging to 3 TAS families (TAS5, TAS9 and TAS10) were identified, 34 putative phased small RNAs and 740 cis/trans‐natural antisense small‐interfering RNAs (nat‐siRNAs) were also found in our results which enriched the tomato small RNAs repository. A large number of genes targeted by those miRNAs and siRNAs were predicted to be involved in the chilling injury responsive process and five of them were verified via degradome sequencing. Based on the above results, a regulatory model that comprehensively reveals the relationships between the small RNAs and their targets was set up. This work provides a foundation for further study of the regulation of miRNAs and siRNAs in the plant in response to chilling injury.     

植物小RNA的研究进展

在植物中发现大量内源性的小RNA,它们与真核生物中的内源性的微RNA和外源性的干扰小RNA有类似的性质和功能。本对植物中小RNA分子的分布、作用机制、功能以及信号传导等方面作一概述。     

Small RNA regulation of ovule development in the cotton plant, <Emphasis Type="Italic">G. hirsutum</Emphasis>L

Background  

The involvement of small RNAs in cotton fiber development is under explored. The objective of this work was to directly clone, annotate, and analyze small RNAs of developing ovules to reveal the candidate small interfering RNA/microRNAs involved in cotton ovule and fiber development.     

Effect of siRNA targeted against MKK4 on myostatin-induced downregulation of differentiation marker gene expression

The c-Jun N-terminal kinase (JNK) pathway was reported to be involved in myostatin signaling and MKK4 was suggested as the only upstream kinase for myostatin-induced JNK activation, implying that MKK4 is a suitable target of RNA interference (RNAi) for blocking myostatin activity. The aim of this study was to evaluate the effect of small interfering RNA (siRNA) targeted against MKK4 on myostatin-induced downregulation of differentiation marker gene expression. Real-time quantitative PCR revealed that the level of MKK4 expression was efficiently reduced by MKK4-specific siRNA. Western blot assays showed that knockdown of MKK4 attenuated the myostatin-induced downregulation of MyoD and myogenin expression.     

小RNA分子与精子发生调控

近来研究发现小RNA(small RNAs)可作为转录后及翻译水平上基因表达调节的重要调节因子,利用小RNA来阐明调节精子发生的分子机制取得了显著进展。这些小RNA主要分为3类,即小干扰RNA(siRNA)、微小RNA(miRNA)以及与piwi蛋白相互作用的RNA(piRNA)。在减数分裂和精子发生过程中,小RNA具有多种生物学功能,如利用siRNA体外转染或体内注射来敲低特定基因从而研究该基因在精子发生过程中的作用;miRNA可能参与精子发生中有丝、减数及后减数分裂阶段的基因表达调节;piRNA主要参与调节雄性生殖细胞减数及后减数分裂的过程,在精子发生中起抑制反转录转座子(retrotransposons)的作用。文章对小RNAs合成、作用机制、功能及展望等最新进展进行了综述。