Role of nuclear RNA in regulating chromatin structure and transcription.

The importance of three-dimensional chromatin organisation in genome regulation has never been clearer. But in spite of the enormous technological advances to probe chromatin organisation in vivo, there is still a lack of mechanistic understanding of how such an arrangement is achieved. Here we review emerging evidence pointing to an intriguing role of nuclear RNA in shaping large-scale chromatin structure and regulating genome function. We suggest this role may be achieved through the formation of a dynamic nuclear mesh that can exploit ATP-driven processes and phase separation of RNA-binding proteins to tune its assembly and material properties.

Kaposi's sarcoma-associated herpesvirus ORF57 protein protects viral transcripts from specific nuclear RNA decay pathways by preventing hMTR4 recruitment.

Nuclear RNAs are subject to a number of RNA decay pathways that serve quality control and regulatory functions. As a result, any virus that expresses its genes in the nucleus must have evolved mechanisms that avoid these pathways, but the how viruses evade nuclear RNA decay remains largely unknown. The multifunctional Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 (Mta) protein is required for the nuclear stability of viral transcripts. In the absence of ORF57, we show that viral transcripts are subject to degradation by two specific nuclear RNA decay pathways, PABPN1 and PAPα/γ-mediated RNA decay (PPD) in which decay factors are recruited through poly(A) tails, and an ARS2-mediated RNA decay pathway dependent on the 5' RNA cap. In transcription pulse chase assays, ORF57 appears to act primarily by inhibiting the ARS2-mediated RNA decay pathway. In the context of viral infection in cultured cells, inactivation of both decay pathways by RNAi is necessary for the restoration of ORF57-dependent viral genes produced from an ORF57-null bacmid. Mechanistically, we demonstrate that ORF57 protects viral transcripts by preventing the recruitment of the exosome co-factor hMTR4. In addition, our data suggest that ORF57 recruitment of ALYREF inhibits hMTR4 association with some viral RNAs, whereas other KSHV transcripts are stabilized by ORF57 in an ALYREF-independent fashion. In conclusion, our studies show that KSHV RNAs are subject to nuclear degradation by two specific host pathways, PPD and ARS2-mediated decay, and ORF57 protects viral transcripts from decay by inhibiting hMTR4 recruitment.

Mitochondrial Genes of Dinoflagellates Are Transcribed by a Nuclear-Encoded Single-Subunit RNA Polymerase.

Dinoflagellates are a large group of algae that contribute significantly to marine productivity and are essential photosynthetic symbionts of corals. Although these algae have fully-functioning mitochondria and chloroplasts, both their organelle genomes have been highly reduced and the genes fragmented and rearranged, with many aberrant transcripts. However, nothing is known about their RNA polymerases. We cloned and sequenced the gene for the nuclear-encoded mitochondrial polymerase (RpoTm) of the dinoflagellate Heterocapsa triquetra and showed that the protein presequence targeted a GFP construct into yeast mitochondria. The gene belongs to a small gene family, which includes a variety of 3'-truncated copies that may have originated by retroposition. The catalytic C-terminal domain of the protein shares nine conserved sequence blocks with other single-subunit polymerases and is predicted to have the same fold as the human enzyme. However, the N-terminal (promoter binding/transcription initiation) domain is not well-conserved. In conjunction with the degenerate nature of the mitochondrial genome, this suggests a requirement for novel accessory factors to ensure the accurate production of functional mRNAs.

A long nuclear-retained non-coding RNA regulates synaptogenesis by modulating gene expression.

A growing number of long nuclear-retained non-coding RNAs (ncRNAs) have recently been described. However, few functions have been elucidated for these ncRNAs. Here, we have characterized the function of one such ncRNA, identified as metastasis-associated lung adenocarcinoma transcript 1 (Malat1). Malat1 RNA is expressed in numerous tissues and is highly abundant in neurons. It is enriched in nuclear speckles only when RNA polymerase II-dependent transcription is active. Knock-down studies revealed that Malat1 modulates the recruitment of SR family pre-mRNA-splicing factors to the transcription site of a transgene array. DNA microarray analysis in Malat1-depleted neuroblastoma cells indicates that Malat1 controls the expression of genes involved not only in nuclear processes, but also in synapse function. In cultured hippocampal neurons, knock-down of Malat1 decreases synaptic density, whereas its over-expression results in a cell-autonomous increase in synaptic density. Our results suggest that Malat1 regulates synapse formation by modulating the expression of genes involved in synapse formation and/or maintenance.

Large functional range of steady-state levels of nuclear and mitochondrial transcripts coding for the subunits of the human mitochondrial OXPHOS system.

We have measured, by reverse transcription and real-time quantitative PCR, the steady-state levels of the mitochondrial and nuclear transcripts encoding several subunits of the human oxidative phosphorylation (OXPHOS) system, in different normal tissues (muscle, liver, trachea, and kidney) and in cultured cells (normal fibroblasts, 143B osteosarcoma cells, 143B206 rho(0) cells). Five mitochondrial transcripts and nine nuclear transcripts were assessed. The measured amounts of these OXPHOS transcripts in muscle samples corroborated data obtained by others using the serial analysis of gene expression (SAGE) method to appraise gene expression in the same type of tissue. Steady-state levels for all the transcripts were found to range over more than two orders of magnitude. Most of the time, the mitochondrial H-strand transcripts were present at higher levels than the nuclear transcripts. The mitochondrial L-strand transcript ND6 was usually present at a low level. Cultured 143B cells contained significantly reduced amounts of mitochondrial transcripts in comparison with the tissue samples. In 143B206 rho(0) cells, fully depleted of mitochondrial DNA, the levels of nuclear OXPHOS transcripts were not modified in comparison with the parental cells. This observation indicated that nuclear transcription is not coordinated with mitochondrial transcription. We also observed that in the different tissues and cells, there is a transcriptional coregulation of all the investigated nuclear genes. Nuclear OXPHOS gene expression seems to be finely regulated.

Specific telomerase RNA residues distant from the template are essential for telomerase function.

The reverse transcriptase telomerase is a ribonucleoprotein complex that adds telomeric repeats to chromosome ends, using a sequence within its endogenous RNA component as a template. Although templating domains of telomerase RNA have been studied in detail, little is known about the roles of the remaining residues, particularly in yeast. We examined the functions of nontemplate telomerase residues in the telomerase RNA of budding yeast Kluyveromyces lactis. Although approximately half of the RNA residues were dispensable for function, four specific regions were essential for telomerase action in vivo. We analyzed the effects of mutating these regions on in vivo function, in vitro telomerase activity, and telomerase RNP assembly. Deletion of two regions resulted in synthesis of stable RNAs that appeared unable to assemble into a stable RNP. Mutating a region near the 5' end of the RNA allowed RNP assembly but abolished enzymatic activity. Mutations in another specific small region of the RNA led to an inactive telomerase RNP with an altered RNA conformation.

Requirement for intron-encoded U22 small nucleolar RNA in 18S ribosomal RNA maturation.

The nucleoli of vertebrate cells contain a number of small RNAs that are generated by the processing of intron fragments of protein-coding gene transcripts. The host gene (UHG) for intro-encoded human U22 is unusual in that it specifies a polyadenylated but apparently noncoding RNA. Depletion of U22 from Xenopus oocytes by oligonucleotide-directed ribonuclease H targeting prevented the processing of 18S ribosomal RNA (rRNA) at both ends. The appearance of 18S rRNA was restored by injection of in vitro-synthesized U22 RNA. These results identify a cellular function for an intron-encoded small RNA.

Methylated cap structures in eukaryotic RNAs: structure, synthesis and functions.

There are more than twenty capped small nuclear RNAs characterized in eukaryotic cells. All the capped RNAs appear to be involved in the processing of other nuclear premessenger or preribosomal RNAs. These RNAs contain either trimethylguanosine (TMG) cap structure or methylated gamma phosphate (Mppp) cap structure. The TMG capped RNAs are capped with M7G during transcription by RNA polymerase II and trimethylated further post-transcriptionally. The Mppp-capped RNAs are transcribed by RNA polymerase III and also capped post-transcriptionally. The cap structures improve the stability of the RNAs and in some cases TMG cap is required for transport of the ribonucleoproteins from cytoplasm to the nucleus. Where tested, the cap structures were not essential for their function in processing other RNAs.