Purification and characterization of a serine protease (CESP) from mature coconut endosperm

Background

Identifying the endogenous RNA induced silencing complex(RISC)-associated RNAs is essential for understanding the cellular regulatory networks by miRNAs. Recently, isolation of RISC-associated mRNAs using antibody was reported, but their method needs a large amount of initial materials. We tried to improve the protocol and constructed an efficient and convenient system for analyzing miRNA and mRNA contents in RISC.

Findings

With our protocol, it is possible to clone both miRNAs and mRNAs from the endogenous RISC-associated RNAs immunoprecipitated from less than 10⁷ cells, and we show the ability of our system to isolate the particular target mRNAs for a specific miRNA from the RISC-associated mRNAs using well-characterized miR-122 as an example. After introduction of miR-122 into HepG2 cells, we found several cDNA clones that have miR-122 target sequences. Four of these clones that were concentrated in RISC but decreased in total RNA fraction are expected to be miR-122 target candidates. Interestingly, we found substantial amounts of Alu-related sequences, including both free Alu RNA and Alu-embedded mRNA, which might be one of the general targets for miRNA, in the cDNA clones from the RISC-associated mRNAs.

Conclusion

Our method thus enables us to examine not only dynamic changes in miRNA and mRNA contents in RISC but also the relationship of miRNA and target mRNA. We believe that our method can contribute to understanding cellular regulatory networks by miRNAs.     

Deep sequencing discovery and profiling of conserved and novel miRNAs in the ovule of <Emphasis Type="Italic">Ginkgo biloba</Emphasis>

Key message

High-throughput sequencing and subsequent analysis identified multiple miRNAs closely related to ovule, indicating that miRNAs are important in Ginkgo biloba ovule.

Abstract

MicroRNAs (miRNAs) are small, noncoding, regulatory RNAs that play crucial regulatory roles in the process of plant growth and development. However, limited information regarding their functions in gymnosperm reproduction is available. Here, we used high-throughput sequencing combined with computational analysis to identify and characterize miRNAs from ovules of G. biloba, and identified 34 conserved miRNA families and 99 novel miRNAs. The precursor sequences of several of the conserved and novel miRNAs were further validated by RT-PCR and sequencing. Furthermore, we found that some target genes, e.g. MYB, homeodomain-leucine zipper (HD-ZIPIII) and auxin response factor (ARF), may be involved in ovule development, and that the significantly enriched pathways of some miRNA targets were related to plant–pathogen interactions and the biosynthesis of secondary metabolites. Twenty-six conserved miRNA families were found to be expressed in both leaves and ovules, while miRNA156, miRNA164, miRNA167, miRNA169, miRNA172 and miRNA390 were up-regulated in ovules. Thus, multiple miRNAs closely related to G. biloba ovule development were identified, resulting in a greater understanding of the important regulatory functions of miRNAs in plant ovules.

     

Efficient and specific inhibition of plant microRNA function by anti-microRNA oligonucleotides (AMOs) in vitro and in vivo

Key message

Anti-microRNA oligonucleotides (AMOs) are efficient and sequence-specific inhibitors of plant miRNA function both in vitro and in vivo.

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that play critical roles in developmental and physiological processes in plants and animals. Although miRNA knockdown by chemically modified antisense oligonucleotides prevails in animal and therapeutic studies, no such application has ever been reported in plants. Here, we show that sucrose-mediated delivery of 2′-O-methyl (2′-O-Me) anti-miRNA oligonucleotides (AMOs) is an efficient and sequence-specific way of inhibiting plant miRNA activity both in vitro and in vivo. Administration of AMOs to rice protoplasts and intact leaves resulted in efficient inhibition of miRNAs with concurrent de-repression of their target genes. AMOs caused simultaneous inhibition of miRNAs from the same family but exerted negligible effects on miRNAs from different families. In rice seedlings, a single-dose AMO treatment conferred long-lasting miRNA inhibition for at least 7 days. Although simultaneous dysregulation of multiple miRNAs by an AMO-and-miRNA-mimic mixture resulted in severe root defects, the phenotypic effects of individual AMOs and miRNA mimics were negligible, suggesting that those miRNAs function together in regulatory networks to ensure homeostasis. Our results validate the utility of AMOs as an efficient tool for plant miRNA loss-of-function studies in vivo, and this approach may prove to be a highly promising general method for unraveling miRNA-mediated gene-regulatory networks.

     

Pegylated derivatives of recombinant human arginase (rhArg1) for sustained in vivo activity in cancer therapy: preparation, characterization and analysis of their pharmacodynamics in vivo and in vitro and action upon hepatocellular carcinoma cell (HCC)

Background

Recent data show aberrant and altered expression of regulatory noncoding micro (mi) RNAs in prostate cancer (PCa). A large number of miRNAs are encoded in organized intronic clusters within many protein coding genes. While expression profiling studies of miRNAs are common place, little is known about the host gene and their resident miRNAs coordinated expression in PCa cells. Furthermore, whether expression of a subset of miRNAs is distinct in androgen-responsive and androgen-independent cells is not clear. Here we have examined the expression of mature miRNAs of miR 17–92, miR 106b-25 and miR 23b-24 clusters along with their host genes C13orf25, MCM7 and AMPO respectively in PCa cell lines.

Results

The expression profiling of miRNAs and host genes was performed in androgen-sensitive MDA PCa 2b and LNCaP as well as in androgen-refractory PC-3 and DU 145 cell culture models of PCa. No significant correlation between the miRNA expression and the intrinsic hormone-responsive property of PCa cells was observed. Androgen-sensitive MDA PCa 2b cells exhibited the highest level of expression of most miRNAs studied in this report. We found significant expression variations between host genes and their resident miRNAs. The expressions of C13orf25 and miR 17–92 cluster as well as MCM7 and miR 106b-25 cluster did not reveal statistically significant correlation, thus suggesting that host genes and resident miRNAs may be expressed independent of each other.

Conclusion

Our results suggest that miRNA expression profiles may not predict intrinsic hormone-sensitive environment of PCa cells. More importantly, our data indicate the possibility of additional novel mechanisms for intronic miRNA processing in PCa cells.     

Inferring the perturbed microRNA regulatory networks from gene expression data using a network propagation based method

Background

MicroRNAs (miRNAs) are a class of endogenous small regulatory RNAs. Identifications of the dys-regulated or perturbed miRNAs and their key target genes are important for understanding the regulatory networks associated with the studied cellular processes. Several computational methods have been developed to infer the perturbed miRNA regulatory networks by integrating genome-wide gene expression data and sequence-based miRNA-target predictions. However, most of them only use the expression information of the miRNA direct targets, rarely considering the secondary effects of miRNA perturbation on the global gene regulatory networks.

Results

We proposed a network propagation based method to infer the perturbed miRNAs and their key target genes by integrating gene expressions and global gene regulatory network information. The method used random walk with restart in gene regulatory networks to model the network effects of the miRNA perturbation. Then, it evaluated the significance of the correlation between the network effects of the miRNA perturbation and the gene differential expression levels with a forward searching strategy. Results show that our method outperformed several compared methods in rediscovering the experimentally perturbed miRNAs in cancer cell lines. Then, we applied it on a gene expression dataset of colorectal cancer clinical patient samples and inferred the perturbed miRNA regulatory networks of colorectal cancer, including several known oncogenic or tumor-suppressive miRNAs, such as miR-17, miR-26 and miR-145.

Conclusions

Our network propagation based method takes advantage of the network effect of the miRNA perturbation on its target genes. It is a useful approach to infer the perturbed miRNAs and their key target genes associated with the studied biological processes using gene expression data.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-255) contains supplementary material, which is available to authorized users.