The Centrality of RNA

New roles for RNAs in biology continue to emerge, and a glance at the history of RNAs may prepare molecular biologists for future discoveries about these powerful molecules. A striking new role for RNAs is their widespread involvement in the regulation of numerous genes, suggesting that there is much yet to discover about these amazing cellular components

Electron Microscope Studies of Heteroduplex DNA from a Deletion Mutant of Bacteriophage phi X-174

A population of double-stranded replicative form of DNA molecules from bacteriophage phi X-174 carrying a deletion of about 9% of the wild-type DNA has been discovered in a sample cultivated under conditions where the phage lysozyme gene is nonessential. The structures of deleted monomers, dimers, and trimers were studied by the electron microscope heteroduplex method. The dimers and trimers are head-to-tail repeats of the deleted monomers. Some interesting examples of the dynamical phenomenon of branch migration in vitro have been observed in heteroduplexes of deleted dimer and trimer strands with undeleted monomer viral strands from the wild-type phage

A Circuitous Route to Noncoding RNA

Most genetic information is expressed as, and transacted by, proteins. Yet, less than 2% of the human genome actually codes for proteins, prompting a search for functions for the other 98% of the genome, once considered to be mostly “junk DNA.” Transcription is pervasive, however, and high-throughput sequencing has identified tens of thousands of distinct RNAs generated from the non—protein—coding portion of the genome (1). These so-called noncoding RNAs vary in length, but like protein-coding RNAs, appear to be linear molecules with 5′ and 3′ termini, reflecting the defined start and end points of RNA polymerase on the DNA template. But do all RNAs have to be linear

Building Robust Transcriptomes with Master Splicing Factors

Coherent splicing networks arise from many discrete splicing decisions regulated in unison. Here, we examine the properties of robust, context-specific splicing networks. We propose that a subset of key splicing regulators, or “master splicing factors,” respond to environmental cues to establish and maintain tissue transcriptomes during development.United States. Public Health Service (RO1-GM34277)United States. Public Health Service (R01-CA133404)United States. Public Health Service (U54-CA112967)National Cancer Institute (U.S.) (P30-CA14051

Super-Enhancer-Mediated RNA Processing Revealed by Integrative MicroRNA Network Analysis

Super-enhancers are an emerging subclass of regulatory regions controlling cell identity and disease genes. However, their biological function and impact on miRNA networks are unclear. Here, we report that super-enhancers drive the biogenesis of master miRNAs crucial for cell identity by enhancing both transcription and Drosha/DGCR8-mediated primary miRNA (pri-miRNA) processing. Super-enhancers, together with broad H3K4me3 domains, shape a tissue-specific and evolutionarily conserved atlas of miRNA expression and function. CRISPR/Cas9 genomics revealed that super-enhancer constituents act cooperatively and facilitate Drosha/DGCR8 recruitment and pri-miRNA processing to boost cell-specific miRNA production. The BET-bromodomain inhibitor JQ1 preferentially inhibits super-enhancer-directed cotranscriptional pri-miRNA processing. Furthermore, super-enhancers are characterized by pervasive interaction with DGCR8/Drosha and DGCR8/Drosha-regulated mRNA stability control, suggesting unique RNA regulation at super-enhancers. Finally, super-enhancers mark multiple miRNAs associated with cancer hallmarks. This study presents principles underlying miRNA biology in health and disease and an unrecognized higher-order property of super-enhancers in RNA processing beyond transcription. Keywords: microRNA; super-enhancer; broad H3K4me3 domain; Drosha; DGCR8; Brd4; cancerUnited States. Public Health Service (Grant R01-CA133404)National Cancer Institute (U.S.) (Grant P30-CA14051

Quantifying argonaute proteins in and out of GW/P-bodies: Implications in microRNA activities

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.National Institutes of Health (U.S.) (Grant R01-CA133404)National Cancer Institute (U.S.) (Grant P01-CA42063)National Cancer Institute (U.S.) (Grant P30-CA14051

Detained introns are a novel, widespread class of post-transcriptionally spliced introns

Deep sequencing of embryonic stem cell RNA revealed many specific internal introns that are significantly more abundant than the other introns within polyadenylated transcripts; we classified these as “detained” introns (DIs). We identified thousands of DIs, many of which are evolutionarily conserved, in human and mouse cell lines as well as the adult mouse liver. DIs can have half-lives of over an hour yet remain in the nucleus and are not subject to nonsense-mediated decay (NMD). Drug inhibition of Clk, a stress-responsive kinase, triggered rapid splicing changes for a specific subset of DIs; half showed increased splicing, and half showed increased intron detention, altering transcript pools of >300 genes. Srsf4, which undergoes a dramatic phosphorylation shift in response to Clk kinase inhibition, regulates the splicing of some DIs, particularly in genes encoding RNA processing and splicing factors. The splicing of some DIs—including those in Mdm4, a negative regulator of p53—was also altered following DNA damage. After 4 h of Clk inhibition, the expression of >400 genes changed significantly, and almost one-third of these are p53 transcriptional targets. These data suggest a widespread mechanism by which the rate of splicing of DIs contributes to the level of gene expression.National Institutes of Health (U.S.) (Grant R01 GM34277-23)American Cancer Society (Novartis Institutes of Biomedical Research Postdoctoral Research Fellowship)National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051

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

Target specificity of the CRISPR-Cas9 system

The CRISPR-Cas9 system, naturally a defense mechanism in prokaryotes, has been repurposed as an RNA-guided DNA targeting platform. It has been widely used for genome editing and transcriptome modulation, and has shown great promise in correcting mutations in human genetic diseases. Off-target effects are a critical issue for all of these applications. Here we review the current status on the target specificity of the CRISPR-Cas9 system.National Institutes of Health (U.S.) (United States Public Health Service Grant RO1-GM34277)National Institutes of Health (U.S.) (United States Public Health Service Grant R01-CA133404)National Cancer Institute (U.S.) (Grant PO1-CA42063)National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051

Argonaute-Bound Small RNAs from Promoter-Proximal RNA Polymerase II

Argonaute (Ago) proteins mediate posttranscriptional gene repression by binding guide miRNAs to regulate targeted RNAs. To confidently assess Ago-bound small RNAs, we adapted a mouse embryonic stem cell system to express a single epitope-tagged Ago protein family member in an inducible manner. Here, we report the small RNA profile of Ago-deficient cells and show that Ago-dependent stability is a common feature of mammalian miRNAs. Using this criteria and immunopurification, we identified an Ago-dependent class of noncanonical miRNAs derived from protein-coding gene promoters, which we name transcriptional start site miRNAs (TSS-miRNAs). A subset of promoter-proximal RNA polymerase II (RNAPII) complexes produces hairpin RNAs that are processed in a DiGeorge syndrome critical region gene 8 (Dgcr8)/Drosha-independent but Dicer-dependent manner. TSS-miRNA activity is detectable from endogenous levels and following overexpression of mRNA constructs. Finally, we present evidence of differential expression and conservation in humans, suggesting important roles in gene regulation.United States. Public Health Service (grant RO1 GM34277)National Cancer Institute (U.S.) (PO1-CA42063)National Cancer Institute (U.S.) (Koch Institute Support (core) grant P30-CA14051)National Institutes of Health (U.S.). Ruth L. Kirschstein National Research Service Award (F32GM101872)National Institutes of Health (U.S.). Ruth L. Kirschstein National Research Service Award (F32CA139902