RNA Subspecies Research Articles

Single-molecule epitranscriptomic analysis of full-length HIV-1 RNAs reveals functional roles of site-specific m6As

Although the significance of chemical modifications on RNA is acknowledged, the evolutionary benefits and specific roles in human immunodeficiency virus (HIV-1) replication remain elusive. Most studies have provided only population-averaged values of modifications for fragmented RNAs at low resolution and have relied on indirect analyses of phenotypic effects by perturbing host effectors. Here we analysed chemical modifications on HIV-1 RNAs at the full-length, single RNA level and nucleotide resolution using direct RNA sequencing methods. Our data reveal an unexpectedly simple HIV-1 modification landscape, highlighting three predominant N6-methyladenosine (m6A) modifications near the 3′ end. More densely installed in spliced viral messenger RNAs than in genomic RNAs, these m6As play a crucial role in maintaining normal levels of HIV-1 RNA splicing and translation. HIV-1 generates diverse RNA subspecies with distinct m6A ensembles, and maintaining multiple of these m6As on its RNAs provides additional stability and resilience to HIV-1 replication, suggesting an unexplored viral RNA-level evolutionary strategy.

Small RNAs in cell-to-cell communications during bacterial infection.

Intercellular communication is a widespread phenomenon in all domains of life. Bacteria have developed many ways of communicating with one another and with other species, either prokaryotic or eukaryotic. RNA has been a key molecule since the beginning of life on Earth, and is one of the carriers of information. Given the current antibiotic crisis, understanding the way in which pathogens communicate can lead towards improved ways to control infections when antimicrobial therapy is not possible. Different subspecies of RNA, non-coding, and of small size, designated here as ncRNAs, have been in recent years the subject of a great research effort, and results have contributed to a growing field of knowledge. This review focuses on four different aspects of ncRNA involvement in cell-to-cell communications during bacterial infections: pathogen recognition by the host, alteration of host microRNA profiles, production of domestic and secreted forms of ncRNAs and subversion of the host responses. The current review article focuses on the most recent discoveries in the field and gives an integrative idea based on the discussed studies.

The emerging role of microRNAs in cardiovascular disease.

Cardiovascular disease remains the most prevalent cause of human morbidity and mortality in ageing Western societies. Basic and translational scientific efforts have focused on the development and improvement of diagnostic and therapeutic strategies to limit the burden of associated diseases, such as stroke and myocardial infarction, and diabetes mellitus and arterial hypertension. Progress in molecular medicine and biology has unravelled a complex epigenetic and post-transcriptional gene-regulating machinery in humans which may limit disease development. An increasing number of attractive molecular strategies, which use the potential of modulating noncoding RNAs, have surfaced over the last decade. Currently, the most extensively studied gene-regulating RNA subspecies are microRNAs, which have been shown to adjust the translational output of coding transcripts by enforcing their degradation and inhibiting their translation into protein. Key findings indicate that microRNAs act as crucial regulators in the majority of human pathologies. Thus, recent research has focused on detecting and modulating microRNAs for therapeutic and biomarker purposes. This review focuses on main and repeated discoveries regarding the role and the therapeutic and biomarker feasibility of microRNAs during cardiovascular disease development and exacerbation.

Spectral phasor analysis of Pyronin Y labeled RNA microenvironments in living cells

We show that the spectral phasor approach of the fluorescent dye Pyronin Y (PY) can be used to identify specific RNA subspecies of ribonuclear proteins complexes in live cells. We applied spectral phasors to isolate intracellular RNA species with similar spectral properties. We identified at least 4 different PY labeled species in live cells and further spatially mapped their presence at the pixel level. Most notable were transcripts in the nucleoli which were spectrally similar to RNA clusters in the cytoplasm. We propose that these species represent ribosomal RNA and clustered ribonucleoprotein complexes. Further, we observed within this cluster Cajal bodies in the proximity of the nucleolus. In addition, transcripts in the cytoplasm undertook a filamentous morphology composed of multiple puncti structures which individually localized along and close to mitochondria but were distinct from mitochondria.

Activation of alternative Jdp2 promoters and functional protein isoforms in T-cell lymphomas by retroviral insertion mutagenesis

Retroviral insertional mutagenesis has been instrumental for the identification of genes important in cancer development. The molecular mechanisms involved in retroviral-mediated activation of proto-oncogenes influence the distribution of insertions within specific regions during tumorigenesis and hence may point to novel gene structures. From a retroviral tagging screen on tumors of 1767 SL3-3 MLV-infected BALB/c mice, intron 2 of the AP-1 repressor Jdp2 locus was found frequently targeted by proviruses resulting in upregulation of non-canonical RNA subspecies. We identified several promoter regions within 1000 bp upstream of exon 3 that allowed for the production of Jdp2 protein isoforms lacking the histone acetylase inhibitory domain INHAT present in canonical Jdp2. The novel Jdp2 isoforms localized to the nucleus and over-expression in murine fibroblast cells induced cell death similar to canonic Jdp2. When expressed in the context of oncogenic NRAS both full length Jdp2 and the shorter isoforms increased anchorage-independent growth. Our results demonstrate a biological function of Jdp2 lacking the INHAT domain and suggest a post-genomic application for the use of retroviral tagging data in identifying new gene products with a potential role in tumorigenesis.

Regulation of c-myc through intranuclear localization of its RNA subspecies

We used fluorescence in situ hybridization (FISH) to detect c-myc RNA subspecies in human COLO 320DM tumor cells. Although the FISH procedure removed the majority of RNAs from the nucleolus, c-myc RNA continued to be detected in both the nucleoplasm and nucleolus. This finding suggests stable association between c-myc RNA and the nucleolus. Nucleolar accumulation of c-myc RNA appeared to be temporally regulated by cell-cycle progression. Hybridization with exon- and strand-specific RNA probes indicated that the non-protein coding exon 1 plays a novel role in determining the subnuclear localization of c-myc RNA. Antisense RNA targeting exon 2 localized only with nucleoplasmic foci, where it might interact with the sense strand. Thus, c-myc gene expression may be regulated by intranuclear localization of its RNA.

Transcript cleavage, attenuation, and an internal promoter in the Rhodobacter capsulatus puc operon.

The stoichiometry of the structural proteins of the photosynthetic apparatus in purple photosynthetic bacteria is achieved primarily by complex regulation of the levels of mRNA encoding the different proteins, which has been studied in the greatest detail in the puf operon. Here we investigated the transcriptional and posttranscriptional regulation of the puc operon, which encodes the peripheral light harvesting complex LHII. We show that, analogous to the puf operon, a primary transcript encoding five puc genes is rapidly processed to generate more stable RNA subspecies. Contrary to previous hypotheses, translational coupling and regulation of puc transcription by puc gene products were found not to occur. A putative RNA stem-loop structure appears to attenuate transcription initiated at the puc operon major promoter. We also found that a minor pucD-internal promoter contributes to the levels of a message that encodes the LHII 14-kDa gamma (PucE) protein.

Characterization of mouse 45S ribosomal RNA subspecies suggests that the first processing cleavage occurs 600 +/- 100 nucleotides from the 5' end and the second 500 +/- 100 nucleotides from the 3' end of a 13.9 kb precursor.

Mouse fibroblasts labeled 1-9 h with 3H-uridine contained radioactive 45S rRNA subspecies of 13.9, 13.3, and 12.8 kb, as determined by hybrid-selection with rDNA plasmids and by electrophoresis in agarose-formaldehyde. The 13.9 kb subspecies contained 5' and 3' terminal rDNA sequences known from the work of Grummt and colleagues to be at or near the ends of the primary transcript. The 13.3 kb subspecies contained the 3' terminal sequence but lacked the 5' terminal sequence. The 12.8 kb subspecies lacked both terminal sequences. Washed nuclei produced one discrete species of 13.9 kb. The results suggested that synthesis of the primary transcript terminated 500 +/- 100 nucleotides beyond the 3' end of 28S rRNA, that the first processing cleavage occurred 600 +/- 100 nucleotides from the origin of synthesis, and the second cleavage occurred near the 3' end of 28S rRNA. Changes in relative radioactivities among the subspecies after serum stimulation or after treatment with low concentrations of cycloheximide suggesting that processing was not perfectly coupled with synthesis and that cycloheximide inhibited one cleavage more than others.

The cryopreservation of mouse leukemic cells. I. Effects on drug resistance and the patterns of nucleic acid metabolism.

Abstract Cultured L1210 lymphocytic leukemic cells resistant to cytosine arabinoside or Cytoxan were frozen under different conditions for up to 5 months and transplanted into recipient mice. Biochemical determinations including DNA, total RNA and drug resistance suggested that the more rapid, less expensive method of freezing mouse leukemic cells enables good retention of each parameter. Examination of cellular and subcellular RNA fingerprints indicated several alterations in the localizations f certain subspecies of RNA. Nonetheless, all cells retained their overall viability and the capacity to induce leukemia in CDF 1 mice.

Isolation and characterization of low molecular weight (4–6S) nuclear RNA in the developing rat brain, and the effect of starvation

(1) Groups of rats were subjected to severe starvation during the development of the foetus and/or during the postnatal period. Nuclear 4–6S RNA isolated from the brains of the 4 groups of rats was studied. (2) On fractionation of the RNA on Sephadex G-100, 3 subspecies of RNA were obtained. One of them had a base composition similar to that of mouse liver t-RNA and showed amino acid acceptor activity. (3) At birth, the total amino acid acceptor activity of 4S RNA obtained from rats born of mothers on a restricted diet was about half the activity of 4S RNA obtained from rats born of mothers on a normal diet. (4) The results indicate that starvation during the development of the foetus and also during the postnatal period causes a lowering in the total body and brain weights; in the total concentration of the low molecular weight (4–6S) RNA; and in the levels of amino acid acceptor activity of 4S RNA in rat brain nuclei. The animals were not capable of returning to the normal metabolic state once damage had been caused by starvation, either during the development of the foetus or in the early postnatal period .

Effect of 3′-deoxyadenosine and 3′-amino-3′-deoxyadenosine on the labelling of RNA sub-species in Ehrlich ascites tumor cells

3′-Deoxyadenosine and 3′-amino-3′-deoxyadenosine exert a differential inhibitory effect on the labelling of nucleochromosomal and cytoplasmic RNA. When these RNA's are further separated into sub-species it is shown that 28-S and 18-S cytoplasmic RNA are very strongly inhibited, whereas 50–60-S nuclear RNA is only slightly inhibited. This 50–60-S nuclear RNA is very rapidly labelled, has a high specific activity, a short half-life and is presumed to serve an intra-nuclear function. A differential inhibition of 32–35-S, 28-S and 18-S RNA, and cytoplasmic 4–5-S RNA is also demonstrated.