Kazimierz T. Tycowski

A Conserved WD40 Protein Binds the Cajal Body Localization Signal of scaRNP Particles

Small Cajal body (CB)-specific RNPs (scaRNPs) function in posttranscriptional modification of small nuclear (sn)RNAs. An RNA element, the CAB box, facilitates CB localization of H/ACA scaRNPs. Using a related element in Drosophila C/D scaRNAs, we purified a fly WD40 repeat protein that UV crosslinks to RNA in a C/D CAB box-dependent manner and associates with C/D and mixed domain C/D-H/ACA scaRNAs. Its human homolog, WDR79, associates with C/D, H/ACA, and mixed domain scaRNAs, as well as with telomerase RNA. WDR79s binding to human H/ACA and mixed domain scaRNAs is CAB box dependent, and its association with mixed domain RNAs also requires the ACA motif, arguing for additional interactions of WDR79 with H/ACA core proteins. We demonstrate a requirement for WDR79 binding in the CB localization of a scaRNA. This and other recent reports establish WDR79 as a central player in the localization and processing of nuclear RNPs.

Formation of triple-helical structures by the 3′-end sequences of MALAT1 and MENβ noncoding RNAs

Stability of the long noncoding-polyadenylated nuclear (PAN) RNA from Kaposis sarcoma-associated herpesvirus is conferred by an expression and nuclear retention element (ENE). The ENE protects PAN RNA from a rapid deadenylation-dependent decay pathway via formation of a triple helix between the U-rich internal loop of the ENE and the 3′-poly(A) tail. Because viruses borrow molecular mechanisms from their hosts, we searched highly abundant human long-noncoding RNAs and identified putative ENE-like structures in metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and multiple endocrine neoplasia-β (MENβ) RNAs. Unlike the PAN ENE, the U-rich internal loops of both predicted cellular ENEs are interrupted by G and C nucleotides and reside upstream of genomically encoded A-rich tracts. We confirmed the ability of MALAT1 and MENβ sequences containing the predicted ENE and A-rich tract to increase the levels of an intronless β-globin reporter RNA. UV thermal denaturation profiles at different pH values support formation of a triple-helical structure composed of multiple U•A-U base triples and a single C•G-C base triple. Additional analyses of the MALAT1 ENE revealed that robust stabilization activity requires an intact triple helix, strong stems at the duplex-triplex junctions, a G-C base pair flanking the triplex to mediate potential A-minor interactions, and the 3′-terminal A of the A-rich tract to form a blunt-ended triplex lacking unpaired nucleotides at the duplex-triplex junction. These examples of triple-helical, ENE-like structures in cellular noncoding RNAs, are unique.

ENHANCED PERSPECTIVE: Small RNA Chaperones for Ribosome Biogenesis

The Howard Hughes Medical Institute, Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06536-0812, USA. A set of small nuclear RNAs of 50 to 200 nucleotides resides in the nucleolus of the cell, the region of the nucleus in which ribosomes are made. J. A. Steitz and K. T. Tycowski discuss what is known about how these RNAs participate in the conversion of the long precursor RNAs to the mature 18S, 5.8S, and 28S rRNAs of the ribosome.

Viral noncoding RNAs: more surprises

Eukaryotic cells produce several classes of long and small noncoding RNA (ncRNA). Many DNA and RNA viruses synthesize their own ncRNAs. Like their host counterparts, viral ncRNAs associate with proteins that are essential for their stability, function, or both. Diverse biological roles--including the regulation of viral replication, viral persistence, host immune evasion, and cellular transformation--have been ascribed to viral ncRNAs. In this review, we focus on the multitude of functions played by ncRNAs produced by animal viruses. We also discuss their biogenesis and mechanisms of action.

Conservation of a Triple-Helix-Forming RNA Stability Element in Noncoding and Genomic RNAs of Diverse Viruses

Abundant expression of the long noncoding (lnc) PAN (polyadenylated nuclear) RNA by the human oncogenic gammaherpesvirus Kaposis sarcoma-associated herpesvirus (KSHV) depends on a cis-element called the expression and nuclear retention element (ENE). The ENE upregulates PAN RNA by inhibiting its rapid nuclear decay through triple-helix formation with the poly(A) tail. Using structure-based bioinformatics, we identified six ENE-like elements in evolutionarily diverse viral genomes. Five are in double-stranded DNA viruses, including mammalian herpesviruses, insect polydnaviruses, and a protist mimivirus. One is in an insect picorna-like positive-strand RNA virus, suggesting that the ENE can counteract cytoplasmic as well as nuclear RNA decay pathways. Functionality of four of the ENEs was demonstrated by increased accumulation of an intronless polyadenylated reporter transcript in human cells. Identification of these ENEs enabled the discovery of PAN RNA homologs in two additional gammaherpesviruses, RRV and EHV2. Our findings demonstrate that searching for structural elements can lead to rapid identification of lncRNAs.

Widespread Inducible Transcription Downstream of Human Genes

Pervasive transcription of the human genome generates RNAs whose mode of formation and functions are largely uncharacterized. Here, we combine RNA-seq with detailed mechanistic studies to describe a transcript type derived from protein-coding genes. The resulting RNAs, which we call DoGs for downstream of gene containing transcripts, possess long non-coding regions (often >45 kb) and remain chromatin bound. DoGs are inducible by osmotic stress through an IP3 receptor signaling-dependent pathway, indicating active regulation. DoG levels are increased by decreased termination of the upstream transcript, a previously undescribed mechanism for rapid transcript induction. Relative depletion of polyA signals in DoG regions correlates with increased levels of DoGs after osmotic stress. We detect DoG transcription in several human cell lines and provide evidence for thousands of DoGs genome wide.

Mapping of ribosomal protein S3 and internally nested snoRNA U15A gene to human chromosome 11q13.3–q13.5

The mammalian ribosome is a massive structure composed of 4 RNA species and about 80 different proteins. One of these ribosomal proteins, S3, appears to function not only in translation but also as an endonuclease in repair of UV-induced DNA damage. Moreover, the first intron of human RPS3 transcripts is processed to generate U15A, a small nucleolar RNA. We localized the nested RPS3/U15A genes to the immediate vicinity of D11S356 and D11S533 on human chromosome 11q13.3-q13.5 using a combination of somatic cell hybrid analysis, fluorescence in situ hybridization, and YAC/STS content mapping. These findings add to the evidence that genes encoding ribosomal proteins are scattered about the human genome.

Abstract IA14: Noncoding RNAs of viral and cellular origin: Links to oncogenesis

EBER2 is a highly abundant nuclear noncoding RNA expressed by Epstein-Barr virus (EBV). Probing its possible chromatin localization by Capture Hybridization Analysis of RNA Targets (CHART) revealed EBER29s presence at the terminal repeats (TRs) of the latent EBV genome, overlapping previously identified binding sites for the important B-cell transcription factor PAX5. EBER2 interacts with and is required for PAX5 localization to the TRs. Knockdown of EBER2 or PAX5 decreases EBV lytic replication, underscoring the essential role of the TRs in viral replication, which is known to be linked to the onset of EBV-associated tumors. Recruitment of the EBER2-PAX5 complex is mediated by base pairing between EBER2 and nascent transcripts from the TR locus. The interaction is evolutionarily conserved in the related primate herpesvirus CeHV15 despite great sequence divergence in both EBER2 and the TRs. Using base pairing with nascent transcripts to guide an interacting transcription factor to its DNA target site in chromatin is a previously undescribed function for a trans-acting noncoding RNA. We have uncovered a novel transcript type derived from human protein-coding genes that is represented by thousands of examples genome-wide. These RNAs, which we call DoGs for downstream of gene containing transcripts, are inducible by osmotic stress through an IP3 receptor signaling-dependent pathway, indicating active regulation. DoGs possess long non-coding regions (often >45 kb) and remain chromatin bound. Their levels are increased by decreased termination of the upstream transcript, a previously undescribed mechanism for rapid transcript induction. Relative depletion of polyA signals in DoG regions correlates with increased levels of DoGs after osmotic stress. DoGs may play a role in reinforcing the nuclear scaffold after stress. 1. Lee, N., Moss, W., Yario, T., Steitz, J.A. (2015). An EBV noncoding RNA binds nascent RNA to drive host PAX5 to viral DNA. Cell 160, 607-618. 2. Vilborg, A., Passarelli, M.C., Yario, T.A., Tycowski, K.T. and Steitz, J.A. (2014). Widespread inducible transcription downstream of human genes. Mol. Cell. 59, 449-461. Citation Format: Joan A. Steitz, Nara Lee, Walter N. Moss, Therese A. Yario, Anna Vilborg, Maria C. Passarelli, Kazimierz T. Tycowski. Noncoding RNAs of viral and cellular origin: Links to oncogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr IA14.