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.     

Systematic evaluation of microRNA processing patterns in tissues, cell lines, and tumors

Very little is known regarding regulation of microRNA (miRNA) biogenesis in normal tissues, tumors, and cell lines. Here, we profiled the expression of 225 precursor and mature miRNAs using real-time PCR and compared the expression levels to determine the processing patterns. RNA from 22 different human tissues, 37 human cancer cell lines, and 16 pancreas and liver tissues/tumors was profiled. The relationship between precursor and mature miRNA expression fell into the following four categories: (1) a direct correlation exists between the precursor and mature miRNA expression in all cells/tissues studied; (2) direct correlation of the precursor and mature miRNA exists, yet the expression is restricted to specific cell lines or tissues; (3) there is detectable expression of mature miRNA in certain cells and tissues while the precursor is expressed in all or most cells/tissues; or (4) both precursor and mature miRNA are not expressed. Pearson correlation between the precursor and mature miRNA expression was closer to one for the tissues but was closer to zero for the cell lines, suggesting that processing of precursor miRNAs is reduced in cancer cell lines. By using Northern blotting, we show that many of these miRNAs (e.g., miR-31, miR-105 and miR-128a) are processed to the precursor, but in situ hybridization analysis demonstrates that these miRNA precursors are retained in the nucleus. We provide a database of the levels of precursor and mature miRNA in a variety of cell types. Our data demonstrate that a large number of miRNAs are transcribed but are not processed to the mature miRNA.     

From microRNAs to targets: pathway discovery in cell fate transitions

MicroRNAs (miRNAs) are 22 nt non-coding RNAs that regulate expression of downstream targets by messenger RNA (mRNA) destabilization and translational inhibition. A large number of eukaryotic mRNAs are targeted by miRNAs, with many individual mRNAs being targeted by multiple miRNAs. Further, a single miRNA can target hundreds of mRNAs, making these small RNAs powerful regulators of cell fate decisions. Such regulation by miRNAs has been observed in the maintenance of the embryonic stem cell (ESC) cell cycle and during ESC differentiation. MiRNAs can also promote the dedifferentiation of somatic cells to induced pluripotent stem cells. During this process they target multiple downstream genes, which represent important nodes of key cellular processes. Here, we review these findings and discuss how miRNAs may be used as tools to discover novel pathways that are involved in cell fate transitions using dedifferentiation of somatic cells to induced pluripotent stem cells as a case study.