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.

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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

The Tap protein mediates the sequence-specific nuclear export of mRNAs bearing the retroviral constitutive transport element (CTE) and also plays a critical role in the sequence nonspecific export of cellular mRNAs. Previously, we have demonstrated that CTE function displays species specificity, that is, the CTE functions in human but not quail cells. Here, we demonstrate that quail Tap fails to support CTE function because it cannot bind the CTE. However, changing a single residue in quail Tap, glutamine 246, to arginine, the residue found in human Tap, rescues both CTE function and CTE binding. This residue, which is located on the exterior of a recently reported molecular structure of Tap, defines a surface on Tap that is critical for CTE binding. These data emphasize the potential importance of cross-species genetic complementation in the identification and characterization of cellular factors that are critical for different aspects of viral replication.

/pdf/using-viral-species-specificity-to-define-a-critical-protein-2cemqfvvqh.pdf

Using viral species specificity to define a critical protein/RNA interaction surface

Sequence requirements for ligand binding and cell surface expression of the Tac antigen, a human interleukin-2 receptor.

Human endogenous retrovirus K (HERV-K) is the name given to an approximately 30-million-year-old family of endogenous retroviruses present at >50 copies per haploid human genome. Previously, the HERV-K were shown to encode a nuclear RNA export factor, termed K-Rev, that is the functional equivalent of the H-Rev protein encoded by human immunodeficiency virus type 1. HERV-K was also shown to contain a cis-acting target element, the HERV-K Rev response element (K-RRE), that allowed the nuclear export of linked RNA transcripts in the presence of either K-Rev or H-Rev. Here, we demonstrate that the functionally defined K-RRE coincides with a statistically highly significant unusual RNA folding region and present a potential RNA secondary structure for the approximately 416-nt K-RRE. Both in vitro and in vivo assays of sequence specific RNA binding were used to map two primary binding sites for K-Rev, and one primary binding site for H-Rev, within the K-RRE. Of note, all three binding sites map to discrete predicted RNA stem-loop subdomains within the larger K-RRE structure. Although almost the entire 416-nt K-RRE was required for the activation of nuclear RNA export in cells expressing K-Rev, mutational inactivation of the binding sites for K-Rev resulted in the selective loss of the K-RRE response to K-Rev but not to H-Rev. Together, these data strongly suggest that the K-RRE, like the H-RRE, coincides with an extensive RNA secondary structure and identify specific sites within the K-RRE that can recruit either K-Rev or H-Rev to HERV-K RNA transcripts.

The human endogenous retrovirus K Rev response element coincides with a predicted RNA folding region.

The emergence of the CRISPR/Cas system of antiviral adaptive immunity in bacteria as a facile system for gene editing in mammalian cells may well lead to gene editing becoming a novel treatment for a range of human diseases, especially those caused by deleterious germline mutations. Another potential target for gene editing are DNA viruses that cause chronic pathogenic diseases that cannot be cured by using currently available drugs. We review the current state of this field and discuss the potential advantages and problems with using a gene editing approach as a treatment for diseases caused by DNA viruses.

Gene Editing: A New Tool for Viral Disease.

A cloned interleukin-2 (IL-2) cDNA was poorly expressed in transfected eukaryotic cells. This low expression was only partly relieved by removal of the homopolymer tail present in the 5′ l...

Bryan R. Cullen

Papers

Using viral species specificity to define a critical protein/RNA interaction surface

Sequence requirements for ligand binding and cell surface expression of the Tac antigen, a human interleukin-2 receptor.

The human endogenous retrovirus K Rev response element coincides with a predicted RNA folding region.

Gene Editing: A New Tool for Viral Disease.

Expression of a Cloned Human Interleukin-2 cDNA Is Enhanced by the Substitution of a Heterologous mRNA Leader Region