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

One of the most profound differences between prokaryotes and eukaryotes is that only the latter segregate RNA synthesis and protein synthesis into distinct cellular compartments, i.e., into the nucleus and cytoplasm, respectively. This segregation offers opportunities for the regulation of both gene expression and RNA posttranscriptional processing that are not available to prokaryotes. However, it also means that eukaryotic cells must continuously transport large numbers of macromolecules between the nucleus and the cytoplasm in order that these can fulfill their appointed role in the life of the cell. In this review, I will describe the mechanisms used by the cell to move different classes of cellular RNA from their nuclear site of synthesis to the cytoplasm, where the majority then participate in the process of protein synthesis. While my primary focus will be on the insights that have been gleaned from the study of nuclear RNA export in vertebrate cells, I will also refer to studies with the yeast Saccharomyces cerevisiae which, because of its genetic malleability, has proven to be a valuable system for the identification of critical components of the nucleocytoplasmic transport machinery. More comprehensive overviews of nucleocytoplasmic transport, particularly of protein import, are provided by several excellent recent reviews in this area (21, 42, 50, 70).

Nuclear RNA Export Pathways

COOH-terminal requirements for the correct processing of a phosphatidylinositol-glycan anchored membrane protein.

The known primate lentiviruses can be divided into two subgroups consisting of the human immunodeficiency virus type 1 (HIV-1) isolates and the related HIV type 2 (HIV-2) and simian immunodeficiency virus (SIV) isolates. HIV-1 has been shown to encode a post-transcriptional trans-activator of viral structural gene expression, termed Rev, that is essential for viral replication in culture. Here, we demonstrate that HIV-2 and SIVmac also encode functional Rev proteins. As in the case of HIV-1, these Rev trans-activators are shown to induce the cytoplasmic expression of the unspliced viral transcripts that encode the viral structural proteins. Unexpectedly, the Rev proteins of HIV-2 and SIVmac proved incapable of activating the cytoplasmic expression of unspliced HIV-1 transcripts, whereas HIV-1 Rev was fully functional in the HIV-2/SIV system. This nonreciprocal complementation may imply a direct role for Rev in mediating the recognition of its viral RNA target sequence.

Functional comparison of the Rev trans-activators encoded by different primate immunodeficiency virus species

It has previously been assumed that the generally high stability of microRNAs (miRNAs) reflects their tight association with Argonaute (Ago) proteins, essential components of the RNA-induced silencing complex (RISC). However, recent data have suggested that the majority of mature miRNAs are not, in fact, Ago associated. Here, we demonstrate that endogenous human miRNAs vary widely, by >100-fold, in their level of RISC association and show that the level of Ago binding is a better indicator of inhibitory potential than is the total level of miRNA expression. While miRNAs of closely similar sequence showed comparable levels of RISC association in the same cell line, these varied between different cell types. Moreover, the level of RISC association could be modulated by overexpression of complementary target mRNAs. Together, these data indicate that the level of RISC association of a given endogenous miRNA is regulated by the available RNA targetome and predicts miRNA function.

Bryan R. Cullen

Papers

Nuclear RNA Export Pathways

COOH-terminal requirements for the correct processing of a phosphatidylinositol-glycan anchored membrane protein.

Functional comparison of the Rev trans-activators encoded by different primate immunodeficiency virus species

Differential RISC association of endogenous human microRNAs predicts their inhibitory potential

Functions of the auxiliary gene products of the human immunodeficiency virus type 1.