David Gatfield

SIRT1 Regulates Circadian Clock Gene Expression through PER2 Deacetylation

The mammalian circadian timing system is composed of a central pacemaker in the suprachiasmatic nucleus of the brain that synchronizes countless subsidiary oscillators in peripheral tissues. The rhythm-generating mechanism is thought to rely on a feedback loop involving positively and negatively acting transcription factors. BMAL1 and CLOCK activate the expression of Period (Per) and Cryptochrome (Cry) genes, and once PER and CRY proteins accumulate to a critical level they form complexes with BMAL1-CLOCK heterodimers and thereby repress the transcription of their own genes. Here, we show that SIRT1, an NAD(+)-dependent protein deacetylase, is required for high-magnitude circadian transcription of several core clock genes, including Bmal1, Rorgamma, Per2, and Cry1. SIRT1 binds CLOCK-BMAL1 in a circadian manner and promotes the deacetylation and degradation of PER2. Given the NAD(+) dependence of SIRT1 deacetylase activity, it is likely that SIRT1 connects cellular metabolism to the circadian core clockwork circuitry.

The exon–exon junction complex provides a binding platform for factors involved in mRNA export and nonsense‐mediated mRNA decay

We recently reported that spliceosomes alter messenger ribonucleoprotein particle (mRNP) composition by depositing several proteins 20–24 nucleotides upstream of mRNA exon–exon junctions. When assembled in vitro, this so‐called ‘exon–exon junction complex’ (EJC) contains at least five proteins: SRm160, DEK, RNPS1, Y14 and REF. To better investigate its functional attributes, we now describe a method for generating spliced mRNAs both in vitro and in vivo that either do or do not carry the EJC. Analysis of these mRNAs in Xenopus laevis oocytes revealed that this complex is the species responsible for enhancing nucleocytoplasmic export of spliced mRNAs. It does so by providing a strong binding site for the mRNA export factors REF and TAP/p15. Moreover, by serving as an anchoring point for the factors Upf2 and Upf3, the EJC provides a direct link between splicing and nonsense‐mediated mRNA decay. Finally, we show that the composition of the EJC is dynamic in vivo and is subject to significant evolution upon mRNA export to the cytoplasm.

Mammalian Genes Are Transcribed with Widely Different Bursting Kinetics

Real-time monitoring of gene expression reveals transcription kinetics of mammalian genes. In prokaryotes and eukaryotes, most genes appear to be transcribed during short periods called transcriptional bursts, interspersed by silent intervals. We describe how such bursts generate gene-specific temporal patterns of messenger RNA (mRNA) synthesis in mammalian cells. To monitor transcription at high temporal resolution, we established various gene trap cell lines and transgenic cell lines expressing a short-lived luciferase protein from an unstable mRNA, and recorded bioluminescence in real time in single cells. Mathematical modeling identified gene-specific on- and off-switching rates in transcriptional activity and mean numbers of mRNAs produced during the bursts. Transcriptional kinetics were markedly altered by cis-regulatory DNA elements. Our analysis demonstrated that bursting kinetics are highly gene-specific, reflecting refractory periods during which genes stay inactive for a certain time before switching on again.

An eIF4AIII-containing complex required for mRNA localization and nonsense-mediated mRNA decay

The specification of both the germ line and abdomen in Drosophila depends on the localization of oskar messenger RNA to the posterior of the oocyte. This localization requires several trans-acting factors, including Barentsz and the Mago–Y14 heterodimer, which assemble with oskar mRNA into ribonucleoprotein particles (RNPs) and localize with it at the posterior pole. Although Barentsz localization in the germ line depends on Mago–Y14, no direct interaction between these proteins has been detected. Here, we demonstrate that the translation initiation factor eIF4AIII interacts with Barentsz and is a component of the oskar messenger RNP localization complex. Moreover, eIF4AIII interacts with Mago–Y14 and thus provides a molecular link between Barentsz and the heterodimer. The mammalian Mago (also known as Magoh)–Y14 heterodimer is a component of the exon junction complex. The exon junction complex is deposited on spliced mRNAs and functions in nonsense-mediated mRNA decay (NMD), a surveillance mechanism that degrades mRNAs with premature translation-termination codons. We show that both Barentsz and eIF4AIII are essential for NMD in human cells. Thus, we have identified eIF4AIII and Barentsz as components of a conserved protein complex that is essential for mRNA localization in flies and NMD in mammals.

Integration of microRNA miR-122 in hepatic circadian gene expression

In liver, most metabolic pathways are under circadian control, and hundreds of protein-encoding genes are thus transcribed in a cyclic fashion. Here we show that rhythmic transcription extends to the locus specifying miR-122, a highly abundant, hepatocyte-specific microRNA. Genetic loss-of-function and gain-of-function experiments have identified the orphan nuclear receptor REV-ERBalpha as the major circadian regulator of mir-122 transcription. Although due to its long half-life mature miR-122 accumulates at nearly constant rates throughout the day, this miRNA is tightly associated with control mechanisms governing circadian gene expression. Thus, the knockdown of miR-122 expression via an antisense oligonucleotide (ASO) strategy resulted in the up- and down-regulation of hundreds of mRNAs, of which a disproportionately high fraction accumulates in a circadian fashion. miR-122 has previously been linked to the regulation of cholesterol and lipid metabolism. The transcripts associated with these pathways indeed show the strongest time point-specific changes upon miR-122 depletion. The identification of Pparbeta/delta and the peroxisome proliferator-activated receptor alpha (PPARalpha) coactivator Smarcd1/Baf60a as novel miR-122 targets suggests an involvement of the circadian metabolic regulators of the PPAR family in miR-122-mediated metabolic control.

REV-ERBα Participates in Circadian SREBP Signaling and Bile Acid Homeostasis

The nuclear receptor REV-ERBα shapes the daily activity profile of Sterol Response Element Binding Protein (SREBP) and thereby participates in the circadian control of cholesterol and bile acid synthesis in the liver.

Nonsense-mediated mRNA decay in Drosophila:at the intersection of the yeast and mammalian pathways

The nonsense‐mediated mRNA decay (NMD) pathway promotes the rapid degradation of mRNAs containing premature stop codons (PTCs). In Caenorhabditis elegans, seven genes (smg1–7) playing an essential role in NMD have been identified. Only SMG2–4 (known as UPF1–3) have orthologs in Saccharomyces cerevisiae. Here we show that the Drosophila orthologs of UPF1–3, SMG1, SMG5 and SMG6 are required for the degradation of PTC‐containing mRNAs, but that there is no SMG7 ortholog in this organism. In contrast, orthologs of SMG5–7 are encoded by the human genome and all three are required for NMD. In human cells, exon boundaries have been shown to play a critical role in defining PTCs. This role is mediated by components of the exon junction complex (EJC). Contrary to expectation, however, we show that the components of the EJC are dispensable for NMD in Drosophila cells. Consistently, PTC definition occurs independently of exon boundaries in Drosophila. Our findings reveal that despite conservation of the NMD machinery, different mechanisms have evolved to discriminate premature from natural stop codons in metazoa.

Nonsense-mediated messenger RNA decay is initiated by endonucleolytic cleavage in Drosophila.

In eukaryotic cells, messenger RNAs harbouring premature termination codons (PTCs) are rapidly degraded by a conserved post-transcriptional mechanism referred to as nonsense-mediated mRNA decay (NMD), which prevents the synthesis of truncated proteins that could be deleterious for the cell. Studies in yeast and mammals indicate that degradation by means of this pathway can occur from both the 5′ end of the message (involving decapping and 5′-to-3′ exonucleolytic digestion by XRN1) or the 3′ end (through accelerated deadenylation and exosome-mediated 3′-to-5′ decay). Here we show that, contrary to expectation, degradation of PTC-containing messages in Drosophila is initiated by endonucleolytic cleavage(s) in the vicinity of the nonsense codon. The resulting 5′ fragment is rapidly degraded by exonucleolytic digestion by the exosome, whereas the 3′ fragment is degraded by XRN1. This decay route is shown for several PTC-containing reporters, as well as an endogenous mRNA that is naturally regulated by NMD. We conclude that, despite conservation in the NMD machinery, PTC-containing transcripts are degraded in Drosophila by a mechanism that differs considerably from those described in yeast and mammals.

The DExH/D box protein HEL/UAP56 is essential for mRNA nuclear export in Drosophila.

Dbp5 is the only member of the DExH/D box family of RNA helicases that is directly implicated in the export of messenger RNAs from the nucleus of yeast and vertebrate cells. Dbp5 localizes in the cytoplasm and at the cytoplasmic face of the nuclear pore complex (NPC). In an attempt to identify proteins present in a highly enriched NPC fraction, two other helicases were detected: RNA helicase A (RHA) and UAP56. This suggested a role for these proteins in nuclear transport. Contrary to expectation, we show that the Drosophila homolog of Dbp5 is not essential for mRNA export in cultured Schneider cells. In contrast, depletion of HEL, the Drosophila homolog of UAP56, inhibits growth and results in a robust accumulation of polyadenylated RNAs within the nucleus. Consequently, incorporation of [35S]methionine into newly synthesized proteins is inhibited. This inhibition affects the expression of both heat-shock and non-heat-shock mRNAs, as well as intron-containing and intronless mRNAs. In HeLa nuclear extracts, UAP56 preferentially, but not exclusively, associates with spliced mRNAs carrying the exon junction complex (EJC). We conclude that HEL is essential for the export of bulk mRNA in Drosophila. The association of human UAP56 with spliced mRNAs suggests that this protein might provide a functional link between splicing and export.

REF1/Aly and the additional exon junction complex proteins are dispensable for nuclear mRNA export

The metazoan proteins UAP56, REF1, and NXF1 are thought to bind sequentially to mRNA to promote its export to the cytoplasm: UAP56 is thought to recruit REF1 to nascent mRNA; REF1 acts as an adaptor protein mediating the association of NXF1 with mRNA, whereas NXF1 translocates the mRNA across the nuclear pore complex. REF1 is a component of the exon–exon junction complex (EJC); thus, the EJC is thought to play a role in the export of spliced mRNA. NXF1 and UAP56 are essential for mRNA export. An essential role for metazoan REF1 or the additional EJC proteins in this process has not been established. Contrary to expectation, we show that REF1 and the additional components of the EJC are dispensable for export of bulk mRNA in Drosophila cells. Only when REF1 and RNPS1 are codepleted, or when all EJC proteins are simultaneously depleted is a partial nuclear accumulation of polyadenylated RNAs observed. Because a significant fraction of bulk mRNA is detected in the cytoplasm of cells depleted of all EJC proteins, we conclude that additional adaptor protein(s) mediate the interaction between NXF1 and cellular mRNAs in metazoa. Our results imply that the essential role of UAP56 in mRNA export is not restricted to the recruitment of REF1.