Specificity, duplex degradation and subcellular localization of antagomirs

MicroRNAs (miRNAs) are an abundant class of 20-23-nt long regulators of gene expression. The study of miRNA function in mice and potential therapeutic approaches largely depend on modified oligonucleotides. We recently demonstrated silencing miRNA function in mice using chemically modified and cholesterol-conjugated RNAs termed 'antagomirs'. Here, we further characterize the properties and function of antagomirs in mice. We demonstrate that antagomirs harbor optimized phosphorothioate modifications, require >19-nt length for highest efficiency and can discriminate between single nucleotide mismatches of the targeted miRNA. Degradation of different chemically protected miRNA/antagomir duplexes in mouse livers and localization of antagomirs in a cytosolic compartment that is distinct from processing (P)-bodies indicates a degradation mechanism independent of the RNA interference (RNAi) pathway. Finally, we show that antagomirs, although incapable of silencing miRNAs in the central nervous system (CNS) when injected systemically, efficiently target miRNAs when injected locally into the mouse cortex. Our data further validate the effectiveness of antagomirs in vivo and should facilitate future studies to silence miRNAs for functional analysis and in clinically relevant settings.

Reliable gene expression measurements from degraded RNA by quantitative real-time PCR depend on short amplicons and a proper normalization

Quantitative reverse transcriptase real-time PCR (QRT-PCR) is a robust method to quantitate RNA abundance. The procedure is highly sensitive and reproducible as long as the initial RNA is intact. However, breaks in the RNA due to chemical or enzymatic cleavage may reduce the number of RNA molecules that contain intact amplicons. As a consequence, the number of molecules available for amplification decreases. We determined the relation between RNA fragmentation and threshold values (Ct values) in subsequent QRT-PCR for four genes in an experimental model of intact and partially hydrolyzed RNA derived from a cell line and we describe the relation between RNA integrity, amplicon size and Ct values in this biologically homogenous system. We demonstrate that degradation-related shifts of Ct values can be compensated by calculating delta Ct values between test genes and the mean values of several control genes. These delta Ct values are less sensitive to fragmentation of the RNA and are unaffected by varying amounts of input RNA. The feasibility of the procedure was demonstrated by comparing Ct values from a larger panel of genes in intact and in partially degraded RNA. We compared Ct values from intact RNA derived from well-preserved tumor material and from fragmented RNA derived from formalin-fixed, paraffin-embedded (FFPE) samples of the same tumors. We demonstrate that the relative abundance of gene expression can be based on FFPE material even when the amount of RNA in the sample and the extent of fragmentation are not known.

THE DOMAINS OF THE CATALYTIC SUBUNIT OF THE EUKARYOTIC RNA DEGRADING EXOSOME, RRP44P, HAVE DISTINCT FUNCTIONS

The exosome is a 3’ to 5’ exoribonuclease complex that consists of ten essential subunits. In the cytoplasm, the exosome degrades mRNA in a general mRNA turnover pathway and in several mRNA surveillance pathways. In the nucleus, the exosome processes RNA precursors to form small, stable, mature RNA species, including rRNA, snRNA, and snoRNA. In addition to processing these RNAs, the nuclear exosome is also involved in degrading aberrantly processed forms of these RNAs, and others, including mRNA. The 3’ to 5’ exoribonuclease activity of the exosome is contributed by the RNB domain of the only catalytically active subunit, Rrp44p, a member of the RNase II family of enzymes. In addition to the RNB domain, Rrp44p consists of three putative RNA binding domains and has an uncharacterized N-terminus, which includes a CR3 region and PIN domain. In an effort to characterize the cellular functions of the domains of Rrp44p, this study identified a second nuclease active site in the PIN domain. Specifically, the PIN domain exhibits endoribonuclease activity in vitro and is essential for exosome function. Further analysis of the nuclease activities of Rrp44p indicate a role for the exoribonuclease activity of Rrp44p in the cytoplasmic and nuclear exosome. This work has also characterized the CR3 region of Rrp44p, a region that has not yet been characterized in any other protein. This region is needed for the majority, if not all, of the cytoplasmic exosome functions as well as for interaction with the exosome. The CR3 region, along with a histidine residue in the N-terminus of Rrp44p, may coordinate a zinc atom. Preliminary evidence supports a role for this coordination in exosome function. Further investigation, however, is needed to determine the molecular dependence of the exosome on the CR3 region of Rrp44p. Despite its initial discovery thirteen years ago, the essential function of Rrp44p, and the exosome, is not yet known. The studies presented here, however, indicate that the essential function of Rrp44p and the exosome is in the nucleus and depends on the nuclease activities of Rrp44p.

Coordinated changes in mRNA turnover, translation, and RNA processing bodies in bronchial epithelial cells following inflammatory stimulation.

Bronchial epithelial cells play a pivotal role in airway inflammation, but little is known about posttranscriptional regulation of mediator gene expression during the inflammatory response in these cells. Here, we show that activation of human bronchial epithelial BEAS-2B cells by proinflammatory cytokines interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-alpha) leads to an increase in the mRNA stability of the key chemokines monocyte chemotactic protein 1 and IL-8, an elevation of the global translation rate, an increase in the levels of several proteins critical for translation, and a reduction of microRNA-mediated translational repression. Moreover, using the BEAS-2B cell system and a mouse model, we found that RNA processing bodies (P bodies), cytoplasmic domains linked to storage and/or degradation of translationally silenced mRNAs, are significantly reduced in activated bronchial epithelial cells, suggesting a physiological role for P bodies in airway inflammation. Our study reveals an orchestrated change among posttranscriptional mechanisms, which help sustain high levels of inflammatory mediator production in bronchial epithelium during the pathogenesis of inflammatory airway diseases.

Messenger RNA half-life measurements in mammalian cells.

The recognition of the importance of mRNA turnover in regulating eukaryotic gene expression has mandated the development of reliable, rigorous, and "user-friendly" methods to accurately measure changes in mRNA stability in mammalian cells. Frequently, mRNA stability is studied indirectly by analyzing the steady-state level of mRNA in the cytoplasm; in this case, changes in mRNA abundance are assumed to reflect only mRNA degradation, an assumption that is not always correct. Although direct measurements of mRNA decay rate can be performed with kinetic labeling techniques and transcriptional inhibitors, these techniques often introduce significant changes in cell physiology. Furthermore, many critical mechanistic issues as to deadenylation kinetics, decay intermediates, and precursor-product relationships cannot be readily addressed by these methods. In light of these concerns, we have previously reported transcriptional pulsing methods based on the c-fos serum-inducible promoter and the tetracycline-regulated (Tet-off) promoter systems to better explain mechanisms of mRNA turnover in mammalian cells. In this chapter, we describe and discuss in detail different protocols that use these two transcriptional pulsing methods. The information described here also provides guidelines to help develop optimal protocols for studying mammalian mRNA turnover in different cell types under a wide range of physiologic conditions.

SMG6 mediated degradation of nonsense mRNA requires phosphorylation-independent interaction with the helicase domain of UPF1

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and eliminated by nonsense-mediated mRNA decay (NMD). NMD targeted mRNAs can be degraded by different routes that all involve phosphorylated UPF1 (P-UPF1) as a starting point. The endonuclease SMG6, which cleaves mRNA near the PTC, is one of three known NMD factors thought to be recruited to nonsense mRNAs by interaction with P-UPF1, leading to eventual mRNA degradation. By MS2-mediated tethering of SMG6 and mutants thereof to a reporter RNA combined with knockdowns of various NMD factors, we demonstrate that besides its endonucleolytic activity, SMG6 also requires UPF1 and SMG1 for inducing RNA decay. Our experiments revealed a phosphorylation-independent interaction between SMG6 and UPF1 that is important for SMG6-mediated mRNA decay and using yeast two hybrid assays, we mapped this interaction to the unique stalk region of the UPF1 helicase domain. This region of UPF1 is essential for SMG6-mediated reporter RNA decay and also for NMD. Our results postulate that besides recruiting SMG6 to its RNA substrates, UPF1 is also required to activate its endonuclease activity.

Nonsense-mediated mRNA decay (NMD) restricts replication of mammalian RNA viruses

A genome-wide siRNA screen against host factors that affect the infection of Semliki Forest virus (SFV), a positive-strand (+)RNA virus, revealed that components of the nonsense-mediated mRNA decay (NMD) pathway restrict early, post-entry steps of the infection cycle. In HeLa cells and primary human fibroblasts, knockdown of UPF1, SMG5 and SMG7 leads to increased levels of viral proteins and RNA and to higher titers of released virus. The inhibitory effect of NMD was stronger when the efficiency of virus replication was impaired by mutations or deletions in the replicase proteins. Accordingly, impairing NMD resulted in a more than 20-fold increased production of these attenuated viruses. Our data suggest that intrinsic features of genomic and sub-genomic viral mRNAs, most likely the extended 3'-UTR length, make them susceptible to NMD. The fact that SFV replication is entirely cytoplasmic strongly suggests that degradation of the viral RNA occurs through the exon junction complex (EJC)-independent mode of NMD. Collectively, our findings uncover a new biological function for NMD as an intrinsic barrier to the translation of early viral proteins and the amplification of (+)RNA viruses in animal cells. Thus, in addition to its role in mRNA surveillance and post-transcriptional gene regulation, NMD also contributes to protect cells from RNA viruses.

SMG6 mediated degradation of nonsense mRNA requires phosphorylation-independent interaction with the helicase domain of UPF1

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and eliminated by nonsense-mediated mRNA decay (NMD). NMD targeted mRNAs can be degraded by different routes that all involve phosphorylated UPF1 (P-UPF1) as a starting point. The endonuclease SMG6, which cleaves mRNA near the PTC, is one of three known NMD factors thought to be recruited to nonsense mRNAs by interaction with P-UPF1, leading to eventual mRNA degradation. By MS2-mediated tethering of SMG6 and mutants thereof to a reporter RNA combined with knockdowns of various NMD factors, we demonstrate that besides its endonucleolytic activity, SMG6 also requires UPF1 and SMG1 for inducing RNA decay. Our experiments revealed a phosphorylation-independent interaction between SMG6 and UPF1 that is important for SMG6-mediated mRNA decay and using yeast two hybrid assays, we mapped this interaction to the unique stalk region of the UPF1 helicase domain. This region of UPF1 is essential for SMG6-mediated reporter RNA decay and also for NMD. Our results postulate that besides recruiting SMG6 to its RNA substrates, UPF1 is also required to activate its endonuclease activity.

Pestiviral E(rns) blocks TLR-3-dependent IFN synthesis by LL37 complexed RNA

The ribonuclease activity of the soluble glycoprotein E(rns) of pestiviruses represents a unique mechanism to circumvent the host's innate immune system by blocking interferon type-I synthesis in response to extracellularly added single- (ss) and double-stranded (ds) RNA. However, the reason why pestiviruses encode a ribonuclease in addition to the abundant serum RNases remained elusive. Here, we show that the 5' UTR and NS5B regions of various strains of the RNA genome of the pestivirus bovine viral diarrhea virus (BVDV) are resistant to serum RNases and are potent TLR-3 agonists. Inhibitory activity of E(rns) was restricted to cleavable RNA products, and did not extend to the synthetic TLR-7/8 agonist R-848. RNA complexed with the antimicrobial peptide LL37 was protected from degradation by E(rns)in vitro but was fully inhibited by E(rns) in its ability to induce IFN in cell cultures, suggesting that the viral protein is mainly active in cleaving RNA in an intracellular compartment. We propose that secreted E(rns) represents a potent IFN antagonist, which degrades viral RNA that is resistant to the ubiquitous host RNases in the extracellular space. Thus, the viral RNase prevents its own pathogen-associated molecular pattern (PAMP) to inadvertently activate the IFN response that might break innate immunotolerance required for persistent pestivirus infections.

RNase E is required for the maturation of ssrA RNA and normal ssrA RNA peptide-tagging activity

During recent studies of ribonucleolytic “degradosome” complexes of Escherichia coli, we found that degradosomes contain certain RNAs as well as RNase E and other protein components. One of these RNAs is ssrA (for small stable RNA) RNA (also known as tm RNA or 10Sa RNA), which functions as both a tRNA and mRNA to tag the C-terminal ends of truncated proteins with a short peptide and target them for degradation. Here, we show that mature 363-nt ssrA RNA is generated by RNase E cleavage at the CCA-3′ terminus of a 457-nt ssrA RNA precursor and that interference with this cleavage in vivo leads to accumulation of the precursor and blockage of SsrA-mediated proteolysis. These results demonstrate that RNase E is required to produce mature ssrA RNA and for normal ssrA RNA peptide-tagging activity. Our findings indicate that RNase E, an enzyme already known to have a central role in RNA processing and decay in E. coli, also has the previously unsuspected ability to affect protein degradation through its role in maturation of the 3′ end of ssrA RNA.

Addition of destabilizing poly(A)-rich sequences to endonuclease cleavage sites during the degradation of chloroplast mRNA

In this work, we report the posttranscriptional addition of poly(A)-rich sequences to mRNA in chloroplasts of higher plants. Several sites in the coding region and the mature end of spinach chloroplast psbA mRNA, which encodes the D1 protein of photosystem II, are detected as polyadenylylated sites. In eukaryotic cells, the addition of multiple adenosine residues to the 3′ end of nuclear RNA plays a key role in generating functional mRNAs and in regulating mRNA degradation. In bacteria, the adenylation of several RNAs greatly accelerates their decay. The poly(A) moiety in the chloroplast, in contrast to that in eukaryotic nuclear encoded and bacterial RNAs, is not a ribohomopolymer of adenosine residues, but clusters of adenosines bounded mostly by guanosines and rarely by cytidines and uridines; it may be as long as several hundred nucleotides. Further analysis of the initial steps of chloroplast psbA mRNA decay revealed specific endonuclease cleavage sites that perfectly matched the sites where poly(A)-rich sequences were added. Our results suggest a mechanism for the degradation of psbA mRNA in which endonucleolytic cleavages are followed by the addition of poly(A)-rich sequences to the upstream cleavage products, which target these RNAs for rapid decay.

Structural origins of the exonuclease resistance of a zwitterionic RNA

Nuclease resistance and RNA affinity are key criteria in the search for optimal antisense nucleic acid modifications, but the origins of the various levels of resistance to nuclease degradation conferred by chemical modification of DNA and RNA are currently not understood. The 2′-O-aminopropyl (AP)-RNA modification displays the highest nuclease resistance among all phosphodiester-based analogues and its RNA binding affinity surpasses that of phosphorothioate DNA by 1°C per modified residue. We found that oligodeoxynucleotides containing AP-RNA residues at their 3′ ends competitively inhibit the degradation of single-stranded DNA by the Escherichia coli Klenow fragment (KF) 3′-5′ exonuclease and snake venom phosphodiesterase. To shed light on the origins of nuclease resistance brought about by the AP modification, we determined the crystal structure of an A-form DNA duplex with AP-RNA modifications at 1.6-Å resolution. In addition, the crystal structures of complexes between short DNA fragments carrying AP-RNA modifications and wild-type KF were determined at resolutions between 2.2 and 3.0 Å and compared with the structure of the complex between oligo(dT) and the D355A/E357A KF mutant. The structural models suggest that interference of the positively charged 2′-O-substituent with the metal ion binding site B of the exonuclease allows AP-RNA to effectively slow down degradation.

Functional topography of nascent RNA in elongation intermediates of RNA polymerase

To determine the dynamics of transcript extrusion from Escherichia coli RNA polymerase (RNAP), we used degradation of the RNA by RNases T1 and A in a series of consecutive elongation complexes (ECs). In intact ECs, even extremely high doses of the RNases were unable to cut the RNA closer than 14–16 nt from the 3′ end. Our results prove that all of the cuts detected within the 14-nt zone are derived from the EC that is denatured during inactivation of the RNases. The protected zone monotonously translocates along the RNA after addition of new nucleotides to the transcript. The upstream region of the RNA heading toward the 5′ end is cleaved and dissociated from the EC, with no effect on the stability and activity of the EC. Most of the current data suggest that an 8- to 10-nt RNA⋅DNA hybrid is formed in the EC. Here, we show that an 8- to 10-nt RNA obtained by truncating the RNase-generated products further with either GreB or pyrophosphate is sufficient for the high stability and activity of the EC. This result suggests that the transcript–RNAP interaction that is required for holding the EC together can be limited to the RNA region involved in the 8- to 10-nt RNA⋅DNA hybrid.