Phillip A. Sharp

RNAi: Double-Stranded RNA Directs the ATP-Dependent Cleavage of mRNA at 21 to 23 Nucleotide Intervals

Double-stranded RNA (dsRNA) directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). Using a recently developed Drosophila in vitro system, we examined the molecular mechanism underlying RNAi. We find that RNAi is ATP dependent yet uncoupled from mRNA translation. During the RNAi reaction, both strands of the dsRNA are processed to RNA segments 21-23 nucleotides in length. Processing of the dsRNA to the small RNA fragments does not require the targeted mRNA. The mRNA is cleaved only within the region of identity with the dsRNA. Cleavage occurs at sites 21-23 nucleotides apart, the same interval observed for the dsRNA itself, suggesting that the 21-23 nucleotide fragments from the dsRNA are guiding mRNA cleavage.

Histone H3K27ac separates active from poised enhancers and predicts developmental state

Developmental programs are controlled by transcription factors and chromatin regulators, which maintain specific gene expression programs through epigenetic modification of the genome. These regulatory events at enhancers contribute to the specific gene expression programs that determine cell state and the potential for differentiation into new cell types. Although enhancer elements are known to be associated with certain histone modifications and transcription factors, the relationship of these modifications to gene expression and developmental state has not been clearly defined. Here we interrogate the epigenetic landscape of enhancer elements in embryonic stem cells and several adult tissues in the mouse. We find that histone H3K27ac distinguishes active enhancers from inactive/poised enhancer elements containing H3K4me1 alone. This indicates that the amount of actively used enhancers is lower than previously anticipated. Furthermore, poised enhancer networks provide clues to unrealized developmental programs. Finally, we show that enhancers are reset during nuclear reprogramming.

Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids

Abstract We have developed a simple and sensitive method for detecting, sizing and mapping RNA transcripts from viral or cloned DNAs. This technique has been used to examine the cytoplasmic transcripts produced during the early phase of adenovirus 2 (Ad2) infection of HeLa cells. Unlabeled total cytoplasmic or oligo (dT)-selected cytoplasmic RNA is hybridized to restriction fragments of 32 P-labeled viral DNA in 80% formamide under conditions above the Tm of the DNA duplex, but below the Tm of the RNA-DNA hybrid duplex (Casey and Davidson, 1977). DNA complements precisely the length of the hybridized RNA are generated by treating with single-strand-specific S1 endonuclease under conditions which do not introduce strand breaks into hybrid duplex. The sizes of the S1-resistant single-stranded DNAs are then determined by alkaline agarose gel electrophoresis (McDonnell, Simon and Studier, 1977). A restriction fragment which terminates within a region coding for an mRNA yields a band equal in size to the portion of the mRNA transcribed from that restriction fragment. This allows unique mapping of coding regions relative to restriction endonuclease cleavage sites. All early Ad2 transcripts are clustered in four regions of the viral DNA. At least five early stable cytoplasmic colinear transcripts are transcribed to the right from the left end of the viral genome, the region of the genome coding functions necessary for transformation of mammalian cells. Transcripts of 650, 350 and 1750 nucleotides map from 1.7 ± 0.5 to 3.6 ± 0.5, 3.6 ± 0.2 to 4.6 ± 0.4, and 4.7 ± 0.3 to 9.5 ± 0.5 units, respectively, and there are two 450 nucleotide transcripts tentatively mapped in the region of 3.0 and 11.0 units. Two overlapping transcripts of 1600 and 1700 nucleotides having the same 3′ terminus and 5′ termini displaced by 100 nucleotides are transcribed to the left from 66.2 ± 0.3 to 61.6 ± 0.2 and 66.5 ± 0.3 to 61.6 ± 0.2 units, respectively. Seven other distinct early stable cytoplasmic transcripts have also been positioned on the genome.

Gene silencing in mammals by small interfering RNAs

Among the 3 billion base pairs of the human genome, there are ∼30,000–40,000 protein-coding genes, but the function of at least half of them remains unknown. A new tool — short interfering RNAs (siRNAs) — has now been developed for systematically deciphering the functions and interactions of these thousands of genes. siRNAs are an intermediate of RNA interference, the process by which double-stranded RNA silences homologous genes. Although the use of siRNAs to silence genes in vertebrate cells was only reported a year ago, the emerging literature indicates that most vertebrate genes can be studied with this technology.

Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells

MicroRNAs (miRNAs) are crucial for normal embryonic stem (ES) cell self-renewal and cellular differentiation, but how miRNA gene expression is controlled by the key transcriptional regulators of ES cells has not been established. We describe here the transcriptional regulatory circuitry of ES cells that incorporates protein-coding and miRNA genes based on high-resolution ChIP-seq data, systematic identification of miRNA promoters, and quantitative sequencing of short transcripts in multiple cell types. We find that the key ES cell transcription factors are associated with promoters for miRNAs that are preferentially expressed in ES cells and with promoters for a set of silent miRNA genes. This silent set of miRNA genes is co-occupied by Polycomb group proteins in ES cells and shows tissue-specific expression in differentiated cells. These data reveal how key ES cell transcription factors promote the ES cell miRNA expression program and integrate miRNAs into the regulatory circuitry controlling ES cell identity.

Targeted Deletion Reveals Essential and Overlapping Functions of the miR-17∼92 Family of miRNA Clusters

miR-17 approximately 92, miR-106b approximately 25, and miR-106a approximately 363 belong to a family of highly conserved miRNA clusters. Amplification and overexpression of miR-1792 is observed in human cancers, and its oncogenic properties have been confirmed in a mouse model of B cell lymphoma. Here we show that mice deficient for miR-17 approximately 92 die shortly after birth with lung hypoplasia and a ventricular septal defect. The miR-17 approximately 92 cluster is also essential for B cell development. Absence of miR-17 approximately 92 leads to increased levels of the proapoptotic protein Bim and inhibits B cell development at the pro-B to pre-B transition. Furthermore, while ablation of miR-106b approximately 25 or miR-106a approximately 363 has no obvious phenotypic consequences, compound mutant embryos lacking both miR-106b approximately 25 and miR-17 approximately 92 die at midgestation. These results provide key insights into the physiologic functions of this family of microRNAs and suggest a link between the oncogenic properties of miR-17 approximately 92 and its functions during B lymphopoiesis and lung development.

Killing the messenger: short RNAs that silence gene expression

Short interfering RNAs can be used to silence gene expression in a sequence-specific manner in a process that is known as RNA interference. The application of RNA interference in mammals has the potential to allow the systematic analysis of gene expression and holds the possibility of therapeutic gene silencing. Much of the promise of RNA interference will depend on the recent advances in short-RNA-based silencing technologies.

Embryonic stem cell-specific MicroRNAs.

We have identified microRNAs (miRNAs) in undifferentiated and differentiated mouse embryonic stem (ES) cells. Some of these appear to be ES cell specific, have related sequences, and are encoded by genomic loci clustered within 2.2 kb of each other. Their expression is repressed as ES cells differentiate into embryoid bodies and is undetectable in adult mouse organs. In contrast, the levels of many previously described miRNAs remain constant or increase upon differentiation. Our results suggest that miRNAs may have a role in the maintenance of the pluripotent cell state and in the regulation of early mammalian development.

Proliferating Cells Express mRNAs with Shortened 3' Untranslated Regions and Fewer MicroRNA Target Sites

Messenger RNA (mRNA) stability, localization, and translation are largely determined by sequences in the 3′ untranslated region (3′UTR). We found a conserved increase in expression of mRNAs terminating at upstream polyadenylation sites after activation of primary murine CD4+ T lymphocytes. This program, resulting in shorter 3′UTRs, is a characteristic of gene expression during immune cell activation and correlates with proliferation across diverse cell types and tissues. Forced expression of full-length 3′UTRs conferred reduced protein expression. In some cases the reduction in protein expression could be reversed by deletion of predicted microRNA target sites in the variably included region. Our data indicate that gene expression is coordinately regulated, such that states of increased proliferation are associated with widespread reductions in the 3′UTR-based regulatory capacity of mRNAs.

c-Myc regulates transcriptional pause release.

Recruitment of the RNA polymerase II (Pol II) transcription initiation apparatus to promoters by specific DNA-binding transcription factors is well recognized as a key regulatory step in gene expression. We report here that promoter-proximal pausing is a general feature of transcription by Pol II in mammalian cells and thus an additional step where regulation of gene expression occurs. This suggests that some transcription factors recruit the transcription apparatus to promoters, whereas others effect promoter-proximal pause release. Indeed, we find that the transcription factor c-Myc, a key regulator of cellular proliferation, plays a major role in Pol II pause release rather than Pol II recruitment at its target genes. We discuss the implications of these results for the role of c-Myc amplification in human cancer.