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

Abstract After viral entry and reverse transcription, HIV-1 proviruses that fail to integrate are epigenetically silenced, but the underlying mechanism has remained unclear. Using a genome-wide CRISPR/Cas9 knockout screen, we identified the host SMC5/6 complex as essential for this epigenetic silencing. We show that SMC5/6 binds to and then SUMOylates unintegrated chromatinized HIV-1 DNA. Inhibition of SUMOylation, either by point mutagenesis of the SMC5/6 component NSMCE2—a SUMO E3 ligase—or using the SUMOylation inhibitor TAK-981, prevents epigenetic silencing, enables transcription from unintegrated HIV-1 DNA and rescues the replication of integrase-deficient HIV-1. Finally, we show that blocking SMC5/6 complex expression, or inhibiting its SUMOylation activity, suppresses the establishment of latent HIV-1 infections in both CD4+ T cell lines and primary human T cells. Collectively, our data show that the SMC5/6 complex plays a direct role in mediating the establishment of HIV-1 latency by epigenetically silencing integration-competent HIV-1 proviruses before integration. 

/pdf/epigenetic-silencing-by-the-smc5-6-complex-mediates-hiv-1-1nsx968i.pdf

Epigenetic silencing by the SMC5/6 complex mediates HIV-1 latency

Transdominant Rev Protein Inhibits Hiv Replication Without Affecting T Cell Function

Publisher Summary Specific ribonucleic acid (RNA)–protein interactions are critical for the posttranscriptional regulation of cellular gene expression at the levels of messenger RNA (mRNA) splicing, nuclear export, translation, and stability and are key to the replication cycle of many pathogenic viruses, including human immunodeficiency virus type 1 (HIV-1), picornaviruses, and influenza viruses. Once a cis -acting RNA regulatory element has been defined, the next step is frequently the identification and subsequent characterization of the cellular or viral protein that determines the phenotype exerted by that element. In addition to the conceptually simple but often technically challenging approach of biochemical purification based on RNA affinity, an in vivo genetic approach can also be used to this problem. However, given that many of the interesting RNA–protein interactions occur in higher eukaryotes and, more specifically, in human cells, it is apparent that a simple and sensitive assay for RNA–protein binding in these same cells would be of considerable utility. The Tat-based assay for analyzing specific RNA–protein interactions relies on the unusual ability of the HIV-1 Tat protein to potently activate transcription from the viral long terminal repeat (LTR) promoter element when targeted to a promoter-proximal RNA target sequence. Specific RNA binding by a Tat-protein Y chimera can in principle be readily detected by cotransfection of an appropriate indicator construct and chimeric Tat protein expression plasmid into a relevant cell line.

[20] Principles and applications of a tat-based assay for analyzing specific RNA-protein interactions in mammalian cells

Our analyses of the sequence of the recombinant virus NTRE7 and comparison with its endogenous and exogenous ALV parents indicate that a section of approximately 400 base pairs at the 3' end of the genome encodes a function which is required for oncogenicity. This section includes an exogenous virus specific region (XSR) of approximately 148 base pairs and the U3 region of the LTR. At present it seems most likely that the function required for oncogenicity is the transcription promoter contained in the U3 region. Assays designed to measure promoter activity of endogenous and exogenous LTRs show a 10 fold difference between the two. This result is consistent with the notion that RAV-0 is non-oncogenic because its promoter activity falls below the limit required for oncogene activation. Our findings suggest that there may be a critical threshold for oncogene activation for which the RAV-2 value represents an upper limit.

Bryan R. Cullen

Papers

Epigenetic silencing by the SMC5/6 complex mediates HIV-1 latency

Transdominant Rev Protein Inhibits Hiv Replication Without Affecting T Cell Function

[20] Principles and applications of a tat-based assay for analyzing specific RNA-protein interactions in mammalian cells

Viral sequences determining the oncogenicity of avian leukosis viruses.

Analysis of viral microRNA expression by elephant endotheliotropic herpesvirus 1.