MicroRNAs: Genomics, Biogenesis, Mechanism, and Function

The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

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Initial sequencing and analysis of the human genome.

抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

http://bartellab.wi.mit.edu/publication_reprints/Bartel_Cell09.pdf

MicroRNAs: Target Recognition and Regulatory Functions

A procedure for extracting plasmid DNA from bacterial cells is described. The method is simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method.

A rapid alkaline extraction procedure for screening recombinant plasmid DNA

MicroRNAs (miRNAs) are a class of ∼22nt non-coding RNAs that regulate the translational potential and stability of mRNAs. Though constituting only 1–4% of human genes, miRNAs are predicted to regulate more than 60% of all mRNAs. The action of miRNAs is mediated through their associations with Argonaute proteins and mRNA targets. Previous studies indicated that though the majority of Argonaute proteins is diffusely distributed in the cytoplasm, a small fraction is consistently observed to be concentrated in a cytoplasmic compartment called GW/P-bodies. In this chapter, we will provide a quantitative and dynamic view of the subcellular localization of miRNA function, followed by a discussion on the possible roles of PBs in miRNA silencing.

Quantifying Argonaute proteins in and out of GW/P-bodies: implications in microRNA activities.

Studies of mammalian RNA interference (RNAi) have focused largely on the actions of microRNAs; however, in other organisms, endogenous short-interfering RNAs (siRNAs) are involved in silencing processes. To date, similar molecules have been difficult to characterize in mammalian cells. P19 is a plant suppressor of RNA silencing that binds with high affinity to siRNAs. Here, the short RNAs bound by P19 in mouse embryonic stem (ES) cells have been characterized. We show that P19 selectively immunoprecipitates endogenous short RNAs from ES cells. Cloning of immunoprecipitated RNA reveals a strong selection for short RNAs that are exact matches to ribosomal RNA (rRNA), with particular short rRNA species highly enriched in P19 immunoprecipitates. Complementary strands to the enriched rRNAs were not cloned, which was surprising because P19 was previously thought to bind only siRNAs. We show that P19 binds tightly to a noncanonical dsRNA substrate comprised of a short RNA annealed to a much longer partner, such that the double-stranded region between the two is 19 base pairs long. Binding to similar endogenous species might explain the association of P19 with short rRNAs in ES cells. Finally, we show that the P19-enriched rRNAs are not involved in canonical RNAi, as they exist in the absence of Dicer and do not function as post-transcriptional gene silencers. Our results support the previous observation that endogenous siRNAs are not abundant molecules in mouse ES cells.

/pdf/characterization-of-the-short-rnas-bound-by-the-p19-36x2lubnlb.pdf

Characterization of the short RNAs bound by the P19 suppressor of RNA silencing in mouse embryonic stem cells.

TFIIB or not TFIIB

A mammalian protein of approximately 220 kDa (p220) was UV-crosslinked to precursor mRNAs (pre-mRNAs) under splicing conditions. The kinetics and biochemical requirements of the UV-crosslinking of p220 corresponded to the kinetics and biochemical requirements of spliceosome formation. On Western blots, antibodies against the yeast splicing factor PRP8 recognized a doublet of proteins, the faster migrating of which comigrated with p220. Furthermore, UV-crosslinked p220 was immunoprecipitated with anti-PRP8 antisera. These results suggest structural conservation of the splicing factor PRP8 from yeast to mammals and show that this protein is in close proximity to the pre-mRNA in the spliceosome.

A mammalian protein of 220 kDa binds pre-mRNAs in the spliceosome: a potential homologue of the yeast PRP8 protein.

Background Hepcidin is an iron regulatory peptide produced by the liver in response to inflammation and elevated systemic iron. Recent studies suggest that circulating monocytes and resident liver macrophages – Kupffer cells – may influence both basal and inflammatory expression of hepcidin.Design and Methods We used an in vitro co-culture model to investigate hepatocyte hepcidin regulation in the presence of activated THP1 macrophages. HuH7 hepatoma cells were co-cultured with differentiated THP1 macrophages for 24 h prior to the measurement of HuH7 hepcidin (HAMP) mRNA expression using quantitative polymerase chain reaction, and HAMP promoter activity using a luciferase reporter assay. Luciferase assays were performed using the wild type HAMP promoter, and constructs containing mutations in BMP/SMAD4, STAT3, C/EBP and E-BOX response elements. Neutralizing antibodies against interleukin-6, interleukin-1β , and the bone morphogenetic protein inhibitor noggin were used to identify the macrophage-derived cytokines involved in the regulation of HAMP expression.Results Co-culturing HuH7 cells with differentiated THP1 cells induced HAMP promoter activity and endogenous HAMP mRNA expression maximally after 24 h. This induction was fully neutralized in the presence of an interleukin-1β antibody, and fully attenuated by mutations of the proximal C/EBP or BMP/SMAD4 response elements.Conclusions Our data suggest that the interleukin-1β and bone morphogenetic protein signaling pathways are central to the regulation of HAMP expression by macrophages in this co-culture model.

/pdf/activated-macrophages-induce-hepcidin-expression-in-huh7-45cpk7cn60.pdf

Phillip A. Sharp

Papers

Quantifying Argonaute proteins in and out of GW/P-bodies: implications in microRNA activities.

Characterization of the short RNAs bound by the P19 suppressor of RNA silencing in mouse embryonic stem cells.

TFIIB or not TFIIB

A mammalian protein of 220 kDa binds pre-mRNAs in the spliceosome: a potential homologue of the yeast PRP8 protein.

Activated macrophages induce hepcidin expression in HuH7 hepatoma cells