The how and why of lncRNA function: An innate immune perspective.

Next-generation sequencing has provided a more complete picture of the composition of the human transcriptome indicating that much of the "blueprint" is a vastness of poorly understood non-protein-coding transcripts. This includes a newly identified class of genes called long noncoding RNAs (lncRNAs). The lack of sequence conservation for lncRNAs across species meant that their biological importance was initially met with some skepticism. LncRNAs mediate their functions through interactions with proteins, RNA, DNA, or a combination of these. Their functions can often be dictated by their localization, sequence, and/or secondary structure. Here we provide a review of the approaches typically adopted to study the complexity of these genes with an emphasis on recent discoveries within the innate immune field. Finally, we discuss the challenges, as well as the emergence of new technologies that will continue to move this field forward and provide greater insight into the biological importance of this class of genes. This article is part of a Special Issue entitled: ncRNA in control of gene expression edited by Kotb Abdelmohsen

The ever-evolving concept of the gene: The use of RNA/Protein experimental techniques to understand genome functions

The completion of the human genome sequence together with advances in sequencing technologies have shifted the paradigm of the genome, as composed of discrete and hereditable coding entities, and have shown the abundance of functional noncoding DNA. This part of the genome, previously dismissed as "junk" DNA, increases proportionally with organismal complexity and contributes to gene regulation beyond the boundaries of known protein-coding genes. Different classes of functionally relevant nonprotein-coding RNAs are transcribed from noncoding DNA sequences. Among them are the long noncoding RNAs (lncRNAs), which are thought to participate in the basal regulation of protein-coding genes at both transcriptional and post-transcriptional levels. Although knowledge of this field is still limited, the ability of lncRNAs to localize in different cellular compartments, to fold into specific secondary structures and to interact with different molecules (RNA or proteins) endows them with multiple regulatory mechanisms. It is becoming evident that lncRNAs may play a crucial role in most biological processes such as the control of development, differentiation and cell growth. This review places the evolution of the concept of the gene in its historical context, from Darwin's hypothetical mechanism of heredity to the post-genomic era. We discuss how the original idea of protein-coding genes as unique determinants of phenotypic traits has been reconsidered in light of the existence of noncoding RNAs. We summarize the technological developments which have been made in the genome-wide identification and study of lncRNAs and emphasize the methodologies that have aided our understanding of the complexity of lncRNA-protein interactions in recent years

A 212-nt long RNA structure in the Tobacco necrosis virus-D RNA genome is resistant to Xrn degradation

Plus-strand RNA viruses can accumulate viral RNA degradation products during infections. Some of these decay intermediates are generated by the cytosolic 5′-to-3′ exoribonuclease Xrn1 (mammals and yeast) or Xrn4 (plants) and are formed when the enzyme stalls on substrate RNAs upon encountering inhibitory RNA structures. Many Xrn-generated RNAs correspond to 3′-terminal segments within the 3′-UTR of viral genomes and perform important functions during infections. Here we have investigated a 3′-terminal small viral RNA (svRNA) generated by Xrn during infections with Tobacco necrosis virus-D (family Tombusviridae). Our results indicate that (i) unlike known stalling RNA structures that are compact and modular, the TNV-D structure encompasses the entire 212 nt of the svRNA and is not functionally transposable, (ii) at least two tertiary interactions within the RNA structure are required for effective Xrn blocking and (iii) most of the svRNA generated in infections is derived from viral polymerase-generated subgenomic mRNA1. In vitro and in vivo analyses allowed for inferences on roles for the svRNA. Our findings provide a new and distinct addition to the growing list of Xrn-resistant viral RNAs and stalling structures found associated with different plant and animal RNA viruses.York University Librarie

Sequence Expression of Supernumerary B Chromosomes: Function or Fluff?

B chromosomes are enigmatic heritable elements found in the genomes of numerous plant and animal species. Contrary to their broad distribution, most B chromosomes are non-essential. For this reason, they are regarded as genome parasites. In order to be stably transmitted through generations, many B chromosomes exhibit the ability to "drive", i.e., they transmit themselves at super-Mendelian frequencies to progeny through directed interactions with the cell division apparatus. To date, very little is understood mechanistically about how B chromosomes drive, although a likely scenario is that expression of B chromosome sequences plays a role. Here, we highlight a handful of previously identified B chromosome sequences, many of which are repetitive and non-coding in nature, that have been shown to be expressed at the transcriptional level. We speculate on how each type of expressed sequence could participate in B chromosome drive based on known functions of RNA in general chromatin- and chromosome-related processes. We also raise some challenges to functionally testing these possible roles, a goal that will be required to more fully understand whether and how B chromosomes interact with components of the cell for drive and transmission

A novel lncRNA as a positive regulator of carotenoid biosynthesis in Fusarium

The fungi Fusarium oxysporum and Fusarium fujikuroi produce carotenoids, lipophilic terpenoid pigments of biotechnological interest, with xanthophyll neurosporaxanthin as the main end product. Their carotenoid biosynthesis is activated by light and negatively regulated by the RING-finger protein CarS. Global transcriptomic analysis identified in both species a putative 1-kb lncRNA that we call carP, referred to as Fo-carP and Ff-carP in each species, upstream to the gene carS and transcribed from the same DNA strand. Fo-carP and Ff-carP are poorly transcribed, but their RNA levels increase in carS mutants. The deletion of Fo-carP or Ff-carP in the respective species results in albino phenotypes, with strong reductions in mRNA levels of structural genes for carotenoid biosynthesis and higher mRNA content of the carS gene, which could explain the low accumulation of carotenoids. Upon alignment, Fo-carP and Ff-carP show 75-80% identity, with short insertions or deletions resulting in a lack of coincident ORFs. Moreover, none of the ORFs found in their sequences have indications of possible coding functions. We conclude that Fo-carP and Ff-carP are regulatory lncRNAs necessary for the active expression of the carotenoid genes in Fusarium through an unknown molecular mechanism, probably related to the control of carS function or expressio

Identification of small RNAs abundant in Burkholderia cenocepacia biofilms reveal putative regulators with a potential role in carbon and iron metabolism

Small RNAs play a regulatory role in many central metabolic processes of bacteria, as well as in developmental processes such as biofilm formation. Small RNAs of Burkholderia cenocepacia, an opportunistic pathogenic beta-proteobacterium, are to date not well characterised. To address that, we performed genome-wide transcriptome structure analysis of biofilm grown B. cenocepacia J2315. 41 unannotated short transcripts were identified in intergenic regions of the B. cenocepacia genome. 15 of these short transcripts, highly abundant in biofilms, widely conserved in Burkholderia sp. and without known function, were selected for in-depth analysis. Expression profiling showed that most of these sRNAs are more abundant in biofilms than in planktonic cultures. Many are also highly abundant in cells grown in minimal media, suggesting they are involved in adaptation to nutrient limitation and growth arrest. Their computationally predicted targets include a high proportion of genes involved in carbon metabolism. Expression and target genes of one sRNA suggest a potential role in regulating iron homoeostasis. The strategy used for this study to detect sRNAs expressed in B. cenocepacia biofilms has successfully identified sRNAs with a regulatory function

Semliki Forest viiruse replikaasivalgu nsP1 uurimine

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Semliki Forest viirus kuulub alfaviiruste perekonda sugukonnas Togaviridae. Alfaviirused on olulised patogeenid ning põhjustavad erinevaid vaevusi nii loomadele kui ka inimestele. Levik looduses toimub sääskede vahendusel. Lisaks tähtsusele patogeensuse tõttu on alfaviirused kasutusel ka bio- ning geenitehnoloogias. Alfaviirustel on positiivse polaarsusega RNA genoom, millelt transleeritakse viiruse replikaasi subühikud (nsP1-nsP4). Antud töö põhiline uurimisobjekt nsP1 vastutab lisaks osalemisele replikatsioonil ja transkriptsioonil replikaasi sidumise eest peremeesraku membraanidele. Antud töö tulemusena tuvastati, et nsP1 palmitüleerimine membraaniga seondumise tugevdamiseks ei ole viirusele hädavajalik. Palmitüleerimine hõlbustab kontaktide loomist nsP1 ja viiruse polümeraasi (nsP4) vahel imetaja rakkudes. Palmitüleerimise puudumisel omandab viirus sekundaarsed mutatsioonid, mis taastavad vastavate kontaktide moodustamise võimet imetajarakkudes. Samad mutatsioonid viiruse elulemust putuka rakkudes olulisel määral ei mõjuta. Leiti, et mittestruktuurse polüproteiini lõikamine nsP1 ja nsP2 vahelt on üks funktsionaalse replikaaskompleksi moodustumise kontrollpunktidest. 1/2 saidi lõikamise kiirendamine vähendab olulisel määral viiruse infektsioonilisust; lõikamise aeglustamisel on väiksem mõju. Mutatsioonanalüüs näitas, et 1/2 saidi lõikust mõjutab eelkõige üks nsP1 valgus lõpus asuv aminohappejääk (P5). Täiendav mutatsioon nsP2 kodeerivas piirkonnas (Q706R) taastas mutantse lõikesaidiga viiruse elujõulisuse ja liitvalgu korrektse lõikamise imetaja rakkudes, ent selline viirus ei suutnud edukalt paljuneda putuka rakkudes ning terviklikku interferoonvastust omavates imetaja rakkudes. Nendest tulemustes lähtuvalt pakuti välja hüpotees, mille kohaselt mutatsioonid 1/2 lõikesaidis ja nsP2 valgus põhjustavad viiruse P123 liitvalgu eluea pikenemise, mis takistab viiruse võimet blokeerida natiivse immuunvastuse teket. Uuritud mutatsioonide bioloogiliste efektide põhjalikumaks uurimiseks on tarvis läbi viia loomkatseid.Semliki Forest virus is a member of the family Togaviridae genus Alpavirus. Alphaviruses are pathogens that cause illness in animals and humans. In nature mosquitos transmit the pathogen. In addition to the importance as pathogens alphaviruses are widely used as biotechnological tools. Alphaviruses have a RNA genome with positive polarity and the replicase subunits (nsP1-nsP4) are translated directly from the genome. nsP1, the protein of investigation in the current thesis, is involved in regulating the formation of a replicase complex and attaching the complex to host cell membranes. In the current thesis it was detected that the palmitoylation of nsP1 as a mean for strengthening the interaction with membranes is not indispensable for the virus. In mammalian cells palmitoylation enables direct contacts to be formed between nsP1 and the viral polymerase. If nsP1 is not palmitoylated, the virus acquires second-site mutations that rescue the ability to form contacts between these proteins. The viability of the virus in insect cells is not much affected by same compensatory mutations. The analysis of the processing requirements between nsP1 and nsP2 in the SFV ns-polyprotein highlighted its importance as one of the major regulatory points in the assembly of a functional replication complex. The accelerated processing of this site severely diminished the infectivity of the corresponding mutant genome, while a reduction in the cleavage efficiency had only a minor effect. The processing of the 1/2 site depends mostly on the amino acid residue in the terminal region of nsP1 (P5). An additional mutation (Q706R) in the nsP2 coding region rescued the viability of the virus with the mutant cleavage site and the correct polyprotein processing. The resulting viruses failed to replicate as efficiently as in mammalian cells in both insect cells and mammalian cells with an intact IFN system. It was suggested that the prolonged stabilty of polyprotein P123 that results from the mutation in the P5 position in the 1/2 cleavage site and the Q706R mutation in the nsP2 coding region contributes to the enhanced native immune response in these cells. Detailed studies using animal models are required to provide further information about the biological implications of these findings

A conformational RNA zipper promotes intron ejection during non-conventional XBP1 mRNA splicing.

The kinase/endonuclease IRE1 is the most conserved signal transducer of the unfolded protein response (UPR), an intracellular signaling network that monitors and regulates the protein folding capacity of the endoplasmic reticulum (ER). Upon sensing protein folding perturbations in the ER, IRE1 initiates the unconventional splicing of XBP1 mRNA culminating in the production of the transcription factor XBP1s, which expands the ER's protein folding capacity. We show that an RNA-intrinsic conformational change causes the intron of XBP1 mRNA to be ejected and the exons to zipper up into an extended stem, juxtaposing the RNA ends for ligation. These conformational rearrangements are important for XBP1 mRNA splicing in vivo. The features that point to such active participation of XBP1 mRNA in the splicing reaction are highly conserved throughout metazoan evolution, supporting their importance in orchestrating XBP1 mRNA processing with efficiency and fidelity

RNA elements directing in vivo assembly of the 7SK/MePCE/Larp7 transcriptional regulatory snRNP

Through controlling the nuclear level of active positive transcription elongation factor b (P-TEFb), the 7SK small nuclear RNA (snRNA) functions as a key regulator of RNA polymerase II transcription. Together with hexamethylene bisacetamide-inducible proteins 1/2 (HEXIM1/2), the 7SK snRNA sequesters P-TEFb into transcriptionally inactive ribonucleoprotein (RNP). In response to transcriptional stimulation, the 7SK/HEXIM/P-TEFb RNP releases P-TEFb to promote polymerase II-mediated messenger RNA synthesis. Besides transiently associating with HEXIM1/2 and P-TEFb, the 7SK snRNA stably interacts with the La-related protein 7 (Larp7) and the methylphosphate capping enzyme (MePCE). In this study, we used in vivo RNA-protein interaction assays to determine the sequence and structural elements of human 7SK snRNA directing assembly of the 7SK/MePCE/Larp7 core snRNP. MePCE interacts with the short 5'-terminal G1-U4/U106-G111 helix-tail motif and Larp7 binds to the 3'-terminal hairpin and the following U-rich tail of 7SK. The overall RNA structure and some particular nucleotides provide the information for specific binding of MePCE and Larp7. We also demonstrate that binding of Larp7 to 7SK is a prerequisite for in vivo recruitment of P-TEFb, indicating that besides providing stability for 7SK, Larp7 directly participates in P-TEFb regulation. Our results provide further explanation for the frequently observed link between Larp7 mutations and cancer development