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

While lentiviral expression vectors are widely used in many facets of molecular biology, due to their ability to stably express heterologous genes in both dividing and non-dividing cells, they suffer from the disadvantage that introns inserted into the vector genome are generally rapidly lost by splicing in packaging cell lines. The presence of an intron, if achievable, has the potential to facilitate the expression of transgene cDNAs, as splicing has been extensively shown to facilitate mRNA biogenesis and function. Moreover, if a stable intron could be introduced into a lentiviral vector, this could greatly facilitate the expression of microRNAs (miRNAs), and especially miRNA clusters, as the introduction of pri-miRNA stems into the exonic region of a lentiviral vector can strongly reduce both vector titer and the expression of any miRNA-linked indicator gene due to cleavage of the vector RNA genome by cellular Drosha. Here, we describe a novel lentiviral vector design in which transgenes and/or miRNAs are expressed using an antisense-orientated, inducible promoter driving an expression cassette bearing a functional intron. We demonstrate that this lentiviral vector, called pTREX, is able to express higher levels of both transgenes and pri-miRNA clusters when compared with a closely similar conventional lentiviral vector.

https://www.tandfonline.com/doi/pdf/10.1080/15476286.2017.1334755

A lentiviral vector bearing a reverse intron demonstrates superior expression of both proteins and microRNAs.

Viral non-coding RNAs (ncRNAs) exist in various forms, many of which appear unique to a particular DNA tumor virus family. In contrast, virally encoded microRNAs (miRNAs) represent a strategy used by several DNA tumor virus families to tap into the same processing and effector machinery utilized by host-derived miRNAs. Whether encoded during latent or lytic infection, ncRNAs are often among the most highly expressed viral transcripts, implying that they have important functions in the viral life cycle. Viral ncRNAs have been shown to contribute to viral gene autoregulation, modification of the host cell apoptotic response, and enhance the translation of viral proteins. While our knowledge of the various viral ncRNAs continues to grow, there remain surprising gaps in our understanding of the functions of some viral ncRNAs, especially given their abundance and the fact that they were discovered decades ago. Here, we provide an overview of the current understanding of the regulatory ncRNAs encoded by the DNA tumor viruses, including the VA RNAs, EBERs, HSURs, PAN, and the recently discovered viral miRNAs.

Non-coding Regulatory RNAs of the DNA Tumor Viruses

Identifying small molecules that selectively bind an RNA target while discriminating against all other cellular RNAs is an important challenge in RNA-targeted drug discovery. Much effort has been directed toward identifying drug-like small molecules that minimize electrostatic and stacking interactions that lead to nonspecific binding of aminoglycosides and intercalators to many stem-loop RNAs. Many such compounds have been reported to bind RNAs and inhibit their cellular activities. However, target engagement and cellular selectivity assays are not routinely performed, and it is often unclear whether functional activity directly results from specific binding to the target RNA. Here, we examined the propensities of three drug-like compounds, previously shown to bind and inhibit the cellular activities of distinct stem-loop RNAs, to bind and inhibit the cellular activities of two unrelated HIV-1 stem-loop RNAs: the transactivation response element (TAR) and the rev response element stem IIB (RREIIB). All compounds bound TAR and RREIIB in vitro, and two inhibited TAR-dependent transactivation and RRE-dependent viral export in cell-based assays while also exhibiting off-target interactions consistent with nonspecific activity. A survey of X-ray and NMR structures of RNA-small molecule complexes revealed that aminoglycosides and drug-like molecules form hydrogen bonds with functional groups commonly accessible in canonical stem-loop RNA motifs, in contrast to ligands that specifically bind riboswitches. Our results demonstrate that drug-like molecules can nonspecifically bind stem-loop RNAs most likely through hydrogen bonding and electrostatic interactions and reinforce the importance of assaying for off-target interactions and RNA selectivity in vitro and in cells when assessing novel RNA-binders.

Understanding the characteristics of nonspecific binding of drug-like compounds to canonical stem-loop RNAs and their implications for functional cellular assays.

Cyclophilin B escorts the hepatitis C virus RNA polymerase: a viral achilles heel?

Novel gene expression indicator genes are provided which encode secretable alkaline phosphatase proteins. By coupling these genes to various expression control systems, it is possible to readily detect gene expression activity. Methods are also provided that can be carried out quickly, without cell lysis or disruption, to detect such activity.

Bryan R. Cullen

Papers

A lentiviral vector bearing a reverse intron demonstrates superior expression of both proteins and microRNAs.

Non-coding Regulatory RNAs of the DNA Tumor Viruses

Understanding the characteristics of nonspecific binding of drug-like compounds to canonical stem-loop RNAs and their implications for functional cellular assays.

Cyclophilin B escorts the hepatitis C virus RNA polymerase: a viral achilles heel?

Secretable forms of alkaline phosphatase