Recent progress in miRNA biogenesis and decay.

MicroRNAs are a class of small regulatory RNAs that mediate regulation of protein synthesis by recognizing sequence elements in mRNAs. MicroRNAs are processed through a series of steps starting from transcription and primary processing in the nucleus to precursor processing and mature function in the cytoplasm. It is also in the cytoplasm where levels of mature microRNAs can be modulated through decay mechanisms. Here, we review the recent progress in the lifetime of a microRNA at all steps required for maintaining their homoeostasis. The increasing knowledge about microRNA regulation upholds great promise as therapeutic targets.

Targeting Polyadenylation for Retention of RNA at Chromatin.

The various steps of RNA polymerase II transcription, including transcription initiation, splicing, and termination, are interlinked and tightly coordinated. Efficient 3'end processing is defined by sequence motifs emerging in the nascent-transcribed RNA strand and the cotranscriptional binding of regulatory proteins. The processing of a mature 3'end consists of cleavage and polyadenylation and is coupled with RNA polymerase II transcription termination and the dissociation of the nascent RNA transcript from the chromatin-associated transcriptional template. The subcellular and subnuclear topological specificity of the various RNA species is important for their functions. For instance, the formation of RNA-binding protein interactions, critical for the final outcome of gene expression, may require the nucleoplasmic fully spliced and polyadenylated form of an RNA transcript. Thus, interfering with the critical step of transcription termination and 3'end formation provides a means for assaying the functional potential of a given RNA of interest.In this protocol, we describe a method for blocking 3'end processing of the nascent RNA transcript, by using RNase H-inactive antisense oligonucleotides targeting cleavage and polyadenylation, delivered via transient transfection in cell culture.