Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction

MicroRNAs: Genomics, Biogenesis, Mechanism, and Function

MicroRNAs (miRNAs) are small RNAs that regulate the expression of complementary messenger RNAs. Hundreds of miRNA genes have been found in diverse animals, and many of these are phylogenetically conserved. With miRNA roles identified in developmental timing, cell death, cell proliferation, haematopoiesis and patterning of the nervous system, evidence is mounting that animal miRNAs are more numerous, and their regulatory impact more pervasive, than was previously suspected.

The functions of animal microRNAs

Recent work has revealed the existence of a class of small non-coding RNA species, known as microRNAs (miRNAs), which have critical functions across various biological processes. Here we use a new, bead-based flow cytometric miRNA expression profiling method to present a systematic expression analysis of 217 mammalian miRNAs from 334 samples, including multiple human cancers. The miRNA profiles are surprisingly informative, reflecting the developmental lineage and differentiation state of the tumours. We observe a general downregulation of miRNAs in tumours compared with normal tissues. Furthermore, we were able to successfully classify poorly differentiated tumours using miRNA expression profiles, whereas messenger RNA profiles were highly inaccurate when applied to the same samples. These findings highlight the potential of miRNA profiling in cancer diagnosis.

/pdf/microrna-expression-profiles-classify-human-cancers-43jqfv3rwu.pdf

MicroRNA expression profiles classify human cancers

MicroRNA (miRNA ) alterations are involved in the initiation and progression of human cancer. The causes of the widespread differential expression of miRNA genes in malignant compared with normal cells can be explained by the location of these genes in cancer-associated genomic regions, by epigenetic mechanisms and by alterations in the miRNA processing machinery. MiRNA-expression profiling of human tumours has identified signatures associated with diagnosis, staging, progression, prognosis and response to treatment. In addition, profiling has been exploited to identify miRNA genes that might represent downstream targets of activated oncogenic pathways, or that target protein- coding genes involved in cancer.

MicroRNA signatures in human cancers

Using a quantitative S1 nuclease protection assay, we demonstrated that acute or chronic infection of avian cells enhances expression of an exogenously introduced rat preproinsulin II gene by approximately equal to 50-fold. The degree of enhancement is shown to vary with the transfection technique used but is independent of the transcription control region of the transfected gene. We conclude that retroviral infection of avian cells enhances expression of transfected DNA in trans by facilitating the uptake of DNA rather than by activating the transfected promoter.

Expression of transfected DNA in avian cells can be enhanced in trans by retroviral infection.

In vivo hypermutation of xenotropic murine leukemia virus-related virus DNA in peripheral blood mononuclear cells of rhesus macaque by APOBEC3 proteins.

Manipulation of host-cell metabolism is an essential aspect of viral replication cycles. Viral co-option of a cellular long non-protein-coding RNA has now been found to be a key step in this process. Manipulation of host-cell metabolism is an essential aspect of viral replication cycles. Viral co-option of a cellular long non-protein-coding RNA has now been found to be a key step in this process.

/pdf/viruses-hijack-a-long-non-coding-rna-9xv9giks8f.pdf

Viruses hijack a long non-coding RNA

Why are humans particularly susceptible to HIV-1, whereas our monkey cousins are impervious to the virus? Part of the answer may lie with the virus' interaction with a cellular system of defense (
 pages 1138–1143
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HIV-1 infection: fooling the gatekeeper

While mammalian somatic cells are incapable of mounting an effective RNA interference (RNAi) response to viral infections, plants and invertebrates are able to generate high levels of viral short interfering RNAs (siRNAs) that can control many infections. In Drosophila, the RNAi response is mediated by the Dicer 2 enzyme (dDcr2) acting in concert with two cofactors called Loqs-PD and R2D2. To examine whether a functional RNAi response could be mounted in human somatic cells, we expressed dDcr2, in the presence or absence of Loqs-PD and/or R2D2, in a previously described human cell line, NoDice/ΔPKR, that lacks functional forms of human Dicer (hDcr) and PKR. We observed significant production of ∼21-nt long siRNAs, derived from a cotransfected double stranded RNA (dsRNA) expression vector, that were loaded into the human RNA-induced silencing complex (RISC) and were able to significantly reduce the expression of a cognate indicator gene. Surprisingly, dDcr2 was able to produce siRNAs even in the absence of Loqs-PD, which is thought to be required for dsRNA cleavage by dDcr2. This result may be explained by our finding that dDcr2 is able to bind the human Loqs-PD homolog TRBP when expressed in human cells in the absence of Loqs-PD. We conclude that it is possible to at least partially rescue the ability of mammalian somatic cells to express functional siRNAs using gene products of invertebrate origin.

/pdf/partial-reconstitution-of-the-rnai-response-in-human-cells-pjj5riv4vy.pdf

Bryan R. Cullen

Papers

Expression of transfected DNA in avian cells can be enhanced in trans by retroviral infection.

In vivo hypermutation of xenotropic murine leukemia virus-related virus DNA in peripheral blood mononuclear cells of rhesus macaque by APOBEC3 proteins.

Viruses hijack a long non-coding RNA

HIV-1 infection: fooling the gatekeeper

Partial reconstitution of the RNAi response in human cells using Drosophila gene products