Highly conserved and cis-acting lncRNAs produced from paralogous regions in the center of HOXA and HOXB clusters in the endoderm lineage.

Long noncoding RNAs (lncRNAs) have been shown to play important roles in gene regulatory networks acting in early development. There has been rapid turnover of lncRNA loci during vertebrate evolution, with few human lncRNAs conserved beyond mammals. The sequences of these rare deeply conserved lncRNAs are typically not similar to each other. Here, we characterize HOXA-AS3 and HOXB-AS3, lncRNAs produced from the central regions of the HOXA and HOXB clusters. Sequence-similar orthologs of both lncRNAs are found in multiple vertebrate species and there is evident sequence similarity between their promoters, suggesting that the production of these lncRNAs predates the duplication of the HOX clusters at the root of the vertebrate lineage. This conservation extends to similar expression patterns of the two lncRNAs, in particular in cells transiently arising during early development or in the adult colon. Functionally, the RNA products of HOXA-AS3 and HOXB-AS3 regulate the expression of their overlapping HOX5-7 genes both in HT-29 cells and during differentiation of human embryonic stem cells. Beyond production of paralogous protein-coding and microRNA genes, the regulatory program in the HOX clusters therefore also relies on paralogous lncRNAs acting in restricted spatial and temporal windows of embryonic development and cell differentiation.

High-resolution mapping of function and protein binding in an RNA nuclear enrichment sequence.

The functions of long RNAs, including mRNAs and long noncoding RNAs (lncRNAs), critically depend on their subcellular localization. The identity of the sequences that dictate subcellular localization and their high-resolution anatomy remain largely unknown. We used a suite of massively parallel RNA assays and libraries containing thousands of sequence variants to pinpoint the functional features within the SIRLOIN element, which dictates nuclear enrichment through hnRNPK recruitment. In addition, we profiled the endogenous SIRLOIN RNA-nucleoprotein complex and identified the nuclear RNA-binding proteins SLTM and SNRNP70 as novel SIRLOIN binders. Taken together, using massively parallel assays, we identified the features that dictate binding of hnRNPK, SLTM, and SNRNP70 to SIRLOIN and found that these factors are jointly required for SIRLOIN activity. Our study thus provides a roadmap for high-throughput dissection of functional sequence elements in long RNAs.

SARS-CoV-2 uses a multipronged strategy to impede host protein synthesis.

The coronavirus SARS-CoV-2 is the cause of the ongoing pandemic of COVID-191. Coronaviruses have developed a variety of mechanisms to repress host mRNA translation to allow the translation of viral mRNA, and concomitantly block the cellular innate immune response2,3. Although several different proteins of SARS-CoV-2 have previously been implicated in shutting off host expression4-7, a comprehensive picture of the effects of SARS-CoV-2 infection on cellular gene expression is lacking. Here we combine RNA sequencing, ribosome profiling and metabolic labelling of newly synthesized RNA to comprehensively define the mechanisms that are used by SARS-CoV-2 to shut off cellular protein synthesis. We show that infection leads to a global reduction in translation, but that viral transcripts are not preferentially translated. Instead, we find that infection leads to the accelerated degradation of cytosolic cellular mRNAs, which facilitates viral takeover of the mRNA pool in infected cells. We reveal that the translation of transcripts that are induced in response to infection (including innate immune genes) is impaired. We demonstrate this impairment is probably mediated by inhibition of nuclear mRNA export, which prevents newly transcribed cellular mRNA from accessing ribosomes. Overall, our results uncover a multipronged strategy that is used by SARS-CoV-2 to take over the translation machinery and to suppress host defences.

Recruitment of the protein phosphatase-1 catalytic subunit to promoters by the dual-function transcription factor RFX1.

RFX proteins are a family of conserved DNA binding proteins involved in various, essential cellular and developmental processes. RFX1 is a ubiquitously expressed, dual-activity transcription factor capable of both activation and repression of target genes. The exact mechanism by which RFX1 regulates its target is not known yet. In this work, we show that the C-terminal repression domain of RFX1 interacts with the Serine/Threonine protein phosphatase PP1c, and that interaction with RFX1 can target PP1c to specific sites in the genome. Given that PP1c was shown to de-phosphorylate several transcription factors, as well as the regulatory C-terminal domain of RNA Polymerase II the recruitment of PP1c to promoters may be a mechanism by which RFX1 regulates the target genes.

Sequences enriched in Alu repeats drive nuclear localization of long RNAs in human cells.

Long noncoding RNAs (lncRNAs) are emerging as key parts of multiple cellular pathways, but their modes of action and how these are dictated by sequence remain unclear. lncRNAs tend to be enriched in the nuclear fraction, whereas most mRNAs are overtly cytoplasmic, although several studies have found that hundreds of mRNAs in various cell types are retained in the nucleus. It is thus conceivable that some mechanisms that promote nuclear enrichment are shared between lncRNAs and mRNAs. Here, to identify elements in lncRNAs and mRNAs that can force nuclear localization, we screened libraries of short fragments tiled across nuclear RNAs, which were cloned into the untranslated regions of an efficiently exported mRNA. The screen identified a short sequence derived from Alu elements and bound by HNRNPK that increased nuclear accumulation. Binding of HNRNPK to C-rich motifs outside Alu elements is also associated with nuclear enrichment in both lncRNAs and mRNAs, and this mechanism is conserved across species. Our results thus identify a pathway for regulation of RNA accumulation and subcellular localization that has been co-opted to regulate the fate of transcripts with integrated Alu elements.

A subset of conserved mammalian long non-coding RNAs are fossils of ancestral protein-coding genes.

BACKGROUND: Only a small portion of human long non-coding RNAs (lncRNAs) appear to be conserved outside of mammals, but the events underlying the birth of new lncRNAs in mammals remain largely unknown. One potential source is remnants of protein-coding genes that transitioned into lncRNAs. RESULTS: We systematically compare lncRNA and protein-coding loci across vertebrates, and estimate that up to 5% of conserved mammalian lncRNAs are derived from lost protein-coding genes. These lncRNAs have specific characteristics, such as broader expression domains, that set them apart from other lncRNAs. Fourteen lncRNAs have sequence similarity with the loci of the contemporary homologs of the lost protein-coding genes. We propose that selection acting on enhancer sequences is mostly responsible for retention of these regions. As an example of an RNA element from a protein-coding ancestor that was retained in the lncRNA, we describe in detail a short translated ORF in the JPX lncRNA that was derived from an upstream ORF in a protein-coding gene and retains some of its functionality. CONCLUSIONS: We estimate that ~ 55 annotated conserved human lncRNAs are derived from parts of ancestral protein-coding genes, and loss of coding potential is thus a non-negligible source of new lncRNAs. Some lncRNAs inherited regulatory elements influencing transcription and translation from their protein-coding ancestors and those elements can influence the expression breadth and functionality of these lncRNAs.

A conserved abundant cytoplasmic long noncoding RNA modulates repression by Pumilio proteins in human cells.

Thousands of long noncoding RNA (lncRNA) genes are encoded in the human genome, and hundreds of them are evolutionarily conserved, but their functions and modes of action remain largely obscure. Particularly enigmatic lncRNAs are those that are exported to the cytoplasm, including NORAD-an abundant and highly conserved cytoplasmic lncRNA. Here we show that most of the sequence of NORAD is comprised of repetitive units that together contain at least 17 functional binding sites for the two mammalian Pumilio homologues. Through binding to PUM1 and PUM2, NORAD modulates the mRNA levels of their targets, which are enriched for genes involved in chromosome segregation during cell division. Our results suggest that some cytoplasmic lncRNAs function by modulating the activities of RNA-binding proteins, an activity which positions them at key junctions of cellular signalling pathways.

Genome-wide chromatin footprinting reveals changes in replication origin architecture induced by pre-RC assembly.

Start sites of DNA replication are marked by the origin recognition complex (ORC), which coordinates Mcm2-7 helicase loading to form the prereplicative complex (pre-RC). Although pre-RC assembly is well characterized in vitro, the process is poorly understood within the local chromatin environment surrounding replication origins. To reveal how the chromatin architecture modulates origin selection and activation, we "footprinted" nucleosomes, transcription factors, and replication proteins at multiple points during the Saccharomyces cerevisiae cell cycle. Our nucleotide-resolution protein occupancy profiles resolved a precise ORC-dependent footprint at 269 origins in G2. A separate class of inefficient origins exhibited protein occupancy only in G1, suggesting that stable ORC chromatin association in G2 is a determinant of origin efficiency. G1 nucleosome remodeling concomitant with pre-RC assembly expanded the origin nucleosome-free region and enhanced activation efficiency. Finally, the local chromatin environment restricts the loading of the Mcm2-7 double hexamer either upstream of or downstream from the ARS consensus sequence (ACS).

DNA replication and transcription programs respond to the same chromatin cues.

DNA replication is a dynamic process that occurs in a temporal order along each of the chromosomes. A consequence of the temporally coordinated activation of replication origins is the establishment of broad domains (>100 kb) that replicate either early or late in S phase. This partitioning of the genome into early and late replication domains is important for maintaining genome stability, gene dosage, and epigenetic inheritance; however, the molecular mechanisms that define and establish these domains are poorly understood. The modENCODE Project provided an opportunity to investigate the chromatin features that define the Drosophila replication timing program in multiple cell lines. The majority of early and late replicating domains in the Drosophila genome were static across all cell lines; however, a small subset of domains was dynamic and exhibited differences in replication timing between the cell lines. Both origin selection and activation contribute to defining the DNA replication program. Our results suggest that static early and late replicating domains were defined at the level of origin selection (ORC binding) and likely mediated by chromatin accessibility. In contrast, dynamic domains exhibited low ORC densities in both cell types, suggesting that origin activation and not origin selection governs the plasticity of the DNA replication program. Finally, we show that the male-specific early replication of the X chromosome is dependent on the dosage compensation complex (DCC), suggesting that the transcription and replication programs respond to the same chromatin cues. Specifically, MOF-mediated hyperacetylation of H4K16 on the X chromosome promotes both the up-regulation of male-specific transcription and origin activation.

Genome-wide localization of replication factors.

Chromatin Immunoprecipitation (ChIP) is a powerful tool for the identification and characterization of protein-DNA interactions in vivo. ChIP has been utilized to study diverse nuclear processes such as transcription regulation, chromatin modification, DNA recombination and DNA replication at specific loci and, more recently, across the entire genome. Advances in genomic approaches, and whole genome sequencing in particular, have made it possible and affordable to comprehensively identify specific protein binding sites throughout the genomes of higher eukaryotes. The dynamic nature of the DNA replication program and the transient occupancy of many replication factors throughout the cell cycle present additional challenges that may not pertain to the mapping of site specific transcription factors. Here we discuss the specific considerations that need to be addressed in the application of ChIP to the genome-wide location analysis of protein factors involved in DNA replication.

Pre-replication complex proteins assemble at regions of low nucleosome occupancy within the Chinese hamster dihydrofolate reductase initiation zone.

Genome-scale mapping of pre-replication complex proteins has not been reported in mammalian cells. Poor enrichment of these proteins at specific sites may be due to dispersed binding, poor epitope availability or cell cycle stage-specific binding. Here, we have mapped sites of biotin-tagged ORC and MCM protein binding in G1-synchronized populations of Chinese hamster cells harboring amplified copies of the dihydrofolate reductase (DHFR) locus, using avidin-affinity purification of biotinylated chromatin followed by high-density microarray analysis across the DHFR locus. We have identified several sites of significant enrichment for both complexes distributed throughout the previously identified initiation zone. Analysis of the frequency of initiations across stretched DNA fibers from the DHFR locus confirmed a broad zone of de-localized initiation activity surrounding the sites of ORC and MCM enrichment. Mapping positions of mononucleosomal DNA empirically and computing nucleosome-positioning information in silico revealed that ORC and MCM map to regions of low measured and predicted nucleosome occupancy. Our results demonstrate that specific sites of ORC and MCM enrichment can be detected within a mammalian initiation zone, and suggest that initiation zones may be regions of generally low nucleosome occupancy where flexible nucleosome positioning permits flexible pre-RC assembly sites.

The "Trojan horse" model-delivery of anti-HBV small interfering RNAs by a recombinant HBV vector.

Hepatitis B virus (HBV) is a small virus that infects the liver. The major obstacle in applying the RNA interference method as an anti-HBV weapon is the challenge to deliver the small interfering RNA molecules to the liver efficiently and specifically. Here we show that HBV-specific short hairpin RNAs (shRNAs) are efficiently expressed from a recombinant HBV into which an shRNA-expressing cassette was inserted, resulting in a significant knock-down of HBV gene expression. Notably, this recombinant HBV still expresses the HBV Core protein, which is targeted by the shRNAs produced by the same vector. Our results set the stage for further use of this recombinant HBV virus with the potential to function as a "Trojan horse"; one that specifically targets the liver and uses the resident virus as an helper for its own propagation, and at the same time eliminate itself and the resident HBV by knocking-down their gene expression.

Autorepression of rfx1 gene expression: functional conservation from yeast to humans in response to DNA replication arrest.

The yeast Saccharomyces cerevisiae Crt1 transcription repressor is an effector of the DNA damage and replication checkpoint pathway. Crt1 binds and represses genes encoding ribonucleotide reductase (RNR) and its own promoter, establishing a negative-feedback pathway. The role of Rfx1, the mammalian Crt1 homologue, remained uncertain. In this study we investigated the possibility that Rfx1 plays a similar function in animal cells. We show here that, like Crt1, Rfx1 binds and represses its own promoter. Furthermore, Rfx1 binding to its promoter is reduced upon induction of a DNA replication block by hydroxyurea, which led to a release of repression. Significantly, like Crt1, Rfx1 binds and represses the RNR-R2 gene. Upon blocking replication and UV treatment, expression of both Rfx1 and RNR-R2 is induced; however, unlike the results seen with the RNR-R2 gene, the derepression of the RFX1 gene is only partially blocked by inhibiting Chk1, the DNA checkpoint kinase. This report provides evidence for a common mechanism for Crt1 and Rfx1 expression and for the conservation of their mode of action in response to a DNA replication block. We suggest that Rfx1 plays a role in the DNA damage response by down-regulating a subset of genes whose expression is increased in response to replication blocking and UV-induced DNA damage.