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

Regulatory pathways governing HIV-1 replication

Charcot-Marie-Tooth disease (CMT1) is the most common form of inherited peripheral neuropathy. Although the disease is genetically heterogeneous, it has been demonstrated that the gene defect is the most frequent type (CMT1A) is the result of a partial duplication of band 17p11.2. Recent studies suggested that the peripheral hypomyelination syndrome in the trembler (Tr) mouse, a possible animal model for CMT1 disease, is associated with a point mutation in the peripheral myelin protein-22 gene (pmp-22). Expression of pmp-22 is particularly high in Schwann cells, and the protein is found in peripheral myelin. We now report that the human PMP-22 gene is contained within the CMT1A duplication. We therefore, suggest that increased dosage of the PMP-22 gene may be the cause of CMT1A neuropathy.

The peripheral myelin protein gene PMP-22 is contained within the Charcot-Marie-Tooth disease type 1A duplication.

Human immunodeficiency virus type 1 (HIV-1) infection of T lymphocytes requires cellular proliferation and DNA synthesis. Human monocytes were shown to have low DNA synthesis rates, yet the monocytotropic BaL isolate of HIV-1 was able to infect these cells efficiently. Monocytes that were irradiated to assure no DNA synthesis could also be readily infected with HIV-1BaL. Such infections were associated with the integration of HIV-1BaL DNA into the high molecular weight, chromosomal DNA of monocytes. Thus, normal, nonproliferating monocytes differ from T lymphocytes in that a productive HIV-1 infection can occur independently of cellular DNA synthesis. These results suggest that normal nonproliferating mononuclear phagocytes, which are relatively resistant to the destructive effects of this virus, may serve as persistent and productive reservoirs for HIV-1 in vivo.

/pdf/productive-human-immunodeficiency-virus-type-1-hiv-1-11xss5isd1.pdf

Productive human immunodeficiency virus type 1 (HIV-1) infection of nonproliferating human monocytes.

Although inhibition of RNA interference (RNAi) by plant virus proteins has been shown to enhance viral replication and pathogenesis in plants, no viral gene product has as yet been shown to inhibit RNAi in vertebrate cells. Here, we present evidence demonstrating that the highly structured ∼160-nucleotide adenoviral VA1 noncoding RNA can inhibit RNAi at physiological levels of expression. VA1, which is expressed at very high levels in adenovirus-infected cells, potently inhibited RNAi induced by short hairpin RNAs (shRNAs) or human microRNA precursors but did not affect RNAi induced by artificial short interfering RNA duplexes. Inhibition appeared to be due both to inhibition of nuclear export of shRNA or premicro-RNA precursors, competition for the Exportin 5 nuclear export factor, and inhibition of Dicer function by direct binding of Dicer. Together, these data argue that adenovirus infection can result in inhibition of RNAi and identify VA1 RNA as the first viral gene product able to inhibit RNAi in human cells.

https://jvi.asm.org/content/jvi/78/23/12868.full.pdf

Adenovirus VA1 noncoding RNA can inhibit small interfering RNA and MicroRNA biogenesis.

Long interspersed element (LINE) 1 retrotransposons are major genomic parasites that represent ≈17% of the human genome. The LINE-1 ORF2 protein is also responsible for the mobility of Alu elements, which constitute a further ≈11% of genomic DNA. Representative members of each element class remain mobile, and deleterious retrotransposition events can induce spontaneous genetic diseases. Here, we demonstrate that APOBEC3A and APOBEC3B, two members of the APOBEC3 family of human innate antiretroviral resistance factors, can enter the nucleus, where LINE-1 and Alu reverse transcription occurs, and specifically inhibit both LINE-1 and Alu retrotransposition. These data suggest that the APOBEC3 protein family may have evolved, at least in part, to defend the integrity of the human genome against endogenous retrotransposons.

https://www.pnas.org/content/pnas/103/23/8780.full.pdf

Bryan R. Cullen

Papers

Regulatory pathways governing HIV-1 replication

The peripheral myelin protein gene PMP-22 is contained within the Charcot-Marie-Tooth disease type 1A duplication.

Productive human immunodeficiency virus type 1 (HIV-1) infection of nonproliferating human monocytes.

Adenovirus VA1 noncoding RNA can inhibit small interfering RNA and MicroRNA biogenesis.

Cellular inhibitors of long interspersed element 1 and Alu retrotransposition.