Jesse R. Zamudio

LincRNA-p21 activates p21 in cis to promote Polycomb target gene expression and to enforce the G1/S checkpoint

The p53-regulated long noncoding RNA lincRNA-p21 has been proposed to act in trans via several mechanisms ranging from repressing genes in the p53 transcriptional network to regulating mRNA translation and protein stability. To further examine lincRNA-p21 function, we generated a conditional knockout mouse model. We find that lincRNA-p21 predominantly functions in cis to activate expression of its neighboring gene, p21. Mechanistically, we show that lincRNA-p21 acts in concert with hnRNP-K as a coactivator for p53-dependent p21 transcription. Additional phenotypes of lincRNA-p21 deficiency could be attributed to diminished p21 levels, including deregulated expression and altered chromatin state of some Polycomb target genes, a defective G1/S checkpoint, increased proliferation rates, and enhanced reprogramming efficiency. These findings indicate that lincRNA-p21 affects global gene expression and influences the p53 tumor suppressor pathway by acting in cis as a locus-restricted coactivator for p53-mediated p21 expression.

Antisense RNA polymerase II divergent transcripts are P-TEFb dependent and substrates for the RNA exosome.

Divergent transcription occurs at the majority of RNA polymerase II (RNAPII) promoters in mouse embryonic stem cells (mESCs), and this activity correlates with CpG islands. Here we report the characterization of upstream antisense transcription in regions encoding transcription start site associated RNAs (TSSa-RNAs) at four divergent CpG island promoters: Isg20l1, Tcea1, Txn1, and Sf3b1. We find that upstream antisense RNAs (uaRNAs) have distinct capped 5′ termini and heterogeneous nonpolyadenylated 3′ ends. uaRNAs are short-lived with average half-lives of 18 minutes and are present at 1–4 copies per cell, approximately one RNA per DNA template. Exosome depletion stabilizes uaRNAs. These uaRNAs are probably initiation products because their capped termini correlate with peaks of paused RNAPII. The pausing factors NELF and DSIF are associated with these antisense polymerases and their sense partners. Knockdown of either NELF or DSIF results in an increase in the levels of uaRNAs. Consistent with P-TEFb controlling release from pausing, treatment with its inhibitor, flavopiridol, decreases uaRNA and nascent mRNA transcripts with similar kinetics. Finally, Isg20l1 induction reveals equivalent increases in transcriptional activity in sense and antisense directions. Together these data show divergent polymerases are regulated after P-TEFb recruitment with uaRNA levels controlled by the exosome.

2′-O-ribose methylation of cap2 in human: function and evolution in a horizontally mobile family

The 5′ cap of human messenger RNA consists of an inverted 7-methylguanosine linked to the first transcribed nucleotide by a unique 5′–5′ triphosphate bond followed by 2′-O-ribose methylation of the first and often the second transcribed nucleotides, likely serving to modify efficiency of transcript processing, translation and stability. We report the validation of a human enzyme that methylates the ribose of the second transcribed nucleotide encoded by FTSJD1, henceforth renamed HMTR2 to reflect function. Purified recombinant hMTr2 protein transfers a methyl group from S-adenosylmethionine to the 2′-O-ribose of the second nucleotide of messenger RNA and small nuclear RNA. Neither N7 methylation of the guanosine cap nor 2′-O-ribose methylation of the first transcribed nucleotide are required for hMTr2, but the presence of cap1 methylation increases hMTr2 activity. The hMTr2 protein is distributed throughout the nucleus and cytosol, in contrast to the nuclear hMTr1. The details of how and why specific transcripts undergo modification with these ribose methylations remains to be elucidated. The 2′-O-ribose RNA cap methyltransferases are present in varying combinations in most eukaryotic and many viral genomes. With the capping enzymes in hand their biological purpose can be ascertained.

Hypermethylated cap 4 maximizes Trypanosoma brucei translation.

Through trans‐splicing of a 39‐nt spliced leader (SL) onto each protein‐coding transcript, mature kinetoplastid mRNA acquire a hypermethylated 5′‐cap structure, but its function has been unclear. Gene deletions for three Trypanosoma brucei cap 2′‐O‐ribose methyltransferases, TbMTr1, TbMTr2 and TbMTr3, reveal distinct roles for four 2′‐O‐methylated nucleotides. Elimination of individual gene pairs yields viable cells; however, attempts at double knock‐outs resulted in the generation of a TbMTr2−/−/TbMTr3−/− cell line only. Absence of both kinetoplastid‐specific enzymes in TbMTr2−/−/TbMTr3−/− lines yielded substrate SL RNA and mRNA with cap 1. TbMTr1−/− translation is comparable with wildtype, while cap 3 and cap 4 loss reduced translation rates, exacerbated by the additional loss of cap 2. TbMTr1−/− and TbMTr2−/−/TbMTr3−/− lines grow to lower densities under normal culture conditions relative to wildtype cells, with growth rate differences apparent under low serum conditions. Cell viability may not tolerate delays at both the nucleolar Sm‐independent and nucleoplasmic Sm‐dependent stages of SL RNA maturation combined with reduced rates of translation. A minimal level of mRNA cap ribose methylation is essential for trypanosome viability, providing the first functional role for the cap 4.

Trypanosoma brucei Spliced Leader RNA Maturation by the Cap 1 2′-O-Ribose Methyltransferase and SLA1 H/ACA snoRNA Pseudouridine Synthase Complex

ABSTRACT Kinetoplastid flagellates attach a 39-nucleotide spliced leader (SL) upstream of protein-coding regions in polycistronic RNA precursors through trans splicing. SL modifications include cap 2′-O-ribose methylation of the first four nucleotides and pseudouridine (ψ) formation at uracil 28. In Trypanosoma brucei, TbMTr1 performs 2′-O-ribose methylation of the first transcribed nucleotide, or cap 1. We report the characterization of an SL RNA processing complex with TbMTr1 and the SLA1 H/ACA small nucleolar ribonucleoprotein (snoRNP) particle that guides SL ψ28 formation. TbMTr1 is in a high-molecular-weight complex containing the four conserved core proteins of H/ACA snoRNPs, a kinetoplastid-specific protein designated methyltransferase-associated protein (TbMTAP), and the SLA1 snoRNA. TbMTAP-null lines are viable but have decreased SL RNA processing efficiency in cap methylation, 3′-end maturation, and ψ28 formation. TbMTAP is required for association between TbMTr1 and the SLA1 snoRNP but does not affect U1 small nuclear RNA methylation. A complex methylation profile in the mRNA population of TbMTAP-null lines indicates an additional effect on cap 4 methylations. The TbMTr1 complex specializes the SLA1 H/ACA snoRNP for efficient processing of multiple modifications on the SL RNA substrate.

Complete Cap 4 Formation Is Not Required for Viability in Trypanosoma brucei

ABSTRACT In kinetoplastids spliced leader (SL) RNA is trans-spliced onto the 5′ ends of all nuclear mRNAs, providing a universal exon with a unique cap. Mature SL contains an m7G cap, ribose 2′-O methylations on the first four nucleotides, and base methylations on nucleotides 1 and 4 (AACU). This structure is referred to as cap 4. Mutagenized SL RNAs that exhibit reduced cap 4 are trans-spliced, but these mRNAs do not associate with polysomes, suggesting a direct role in translation for cap 4, the primary SL sequence, or both. To separate SL RNA sequence alterations from cap 4 maturation, we have examined two ribose 2′-O-methyltransferases in Trypanosoma brucei. Both enzymes fall into the Rossmann fold class of methyltransferases and model into a conserved structure based on vaccinia virus homolog VP39. Knockdown of the methyltransferases individually or in combination did not affect growth rates and suggests a temporal placement in the cap 4 formation cascade: TbMT417 modifies A2 and is not required for subsequent steps; TbMT511 methylates C3, without which U4 methylations are reduced. Incomplete cap 4 maturation was reflected in substrate SL and mRNA populations. Recombinant methyltransferases bind to a methyl donor and show preference for m7G-capped RNAs in vitro. Both enzymes reside in the nucleoplasm. Based on the cap phenotype of substrate SL stranded in the cytosol, A2, C3, and U4 methylations are added after nuclear reimport of Sm protein-complexed substrate SL RNA. As mature cap 4 is dispensable for translation, cap 1 modifications and/or SL sequences are implicated in ribosomal interaction.

The 2-O-Ribose Methyltransferase for Cap 1 of Spliced Leader RNA and U1 Small Nuclear RNA in Trypanosoma brucei†

ABSTRACT mRNA cap 1 2′-O-ribose methylation is a widespread modification that is implicated in processing, trafficking, and translational control in eukaryotic systems. The eukaryotic enzyme has yet to be identified. In kinetoplastid flagellates trans-splicing of spliced leader (SL) to polycistronic precursors conveys a hypermethylated cap 4, including a cap 0 m7G and seven additional methylations on the first 4 nucleotides, to all nuclear mRNAs. We report the first eukaryotic cap 1 2′-O-ribose methyltransferase, TbMTr1, a member of a conserved family of viral and eukaryotic enzymes. Recombinant TbMTr1 methylates the ribose of the first nucleotide of an m7G-capped substrate. Knockdowns and null mutants of TbMTr1 in Trypanosoma brucei grow normally, with loss of 2′-O-ribose methylation at cap 1 on substrate SL RNA and U1 small nuclear RNA. TbMTr1-null cells have an accumulation of cap 0 substrate without further methylation, while spliced mRNA is modified efficiently at position 4 in the absence of 2′-O-ribose methylation at position 1; downstream cap 4 methylations are independent of cap 1. Based on TbMTr1-green fluorescent protein localization, 2′-O-ribose methylation at position 1 occurs in the nucleus. Accumulation of 3′-extended SL RNA substrate indicates a delay in processing and suggests a synergistic role for cap 1 in maturation.

The TbMTr1 spliced leader RNA cap 1 2'-O-ribose methyltransferase from Trypanosoma brucei acts with substrate specificity.

In metazoa cap 1 (m7GpppNmp-RNA) is linked to higher levels of translation; however, the enzyme responsible remains unidentified. We have validated the first eukaryotic encoded cap 1 2 ′-O-ribose methyltransferase, TbMTr1, a member of a conserved family that modifies the first transcribed nucleotide of spliced leader and U1 small nuclear RNAs in the kinetoplastid protozoan Trypanosoma brucei. In addition to cap 0 (m7GpppNp-RNA), mRNA in these parasites has ribose methylations on the first four nucleotides with base methylations on the first and fourth (m7Gpppm6,6AmpAmpCmpm3Ump-SL RNA) conveyed via trans-splicing of a universal spliced leader. The function of this cap 4 is unclear. Spliced leader is the majority RNA polymerase II transcript; the RNA polymerase III-transcribed U1 small nuclear RNA has the same first four nucleotides as spliced leader, but it receives an m2,2,7G cap with hypermethylation at position one only (m2,2,7Gpppm6,6AmpApCpUp-U1 snRNA). Here we examine the biochemical properties of recombinant TbMTr1. Active over a pH range of 6.0 to 9.5, TbMTr1 is sensitive to Mg2+. Positions Lys95-Asp204-Lys259-Glu285 constitute the conserved catalytic core. A guanosine cap on RNA independent of its N7 methylation status is required for substrate recognition, but an m2,2,7G-cap is not recognized. TbMTr1 favors the spliced leader 5′ sequence, as reflected by a preference for A at position 1 and modulation of activity for substrates with base changes at positions 2 and 3. With similarities to human cap 1 methyltransferase activity, TbMTr1 is an excellent model for higher eukaryotic cap 1 methyltransferases and the consequences of cap 1 modification.

SL RNA Biogenesis in Kinetoplastids: A Long and Winding Road

The spliced leader (SL) RNA is a defining element in the gene expression of kinetoplastids. The first 39 nt of this small RNA are trans-spliced onto every nuclear message, providing a unique hypermethylated cap and sequence elements required for stability and translation. Transcribed from a large tandem array, the journey that each primary SL transcript takes en route to splicing is marked by molecular modifications. Methylation, pseudouridylation, and 3′-end nuclease processing contribute to the mature product. The consequences of this elaborate pathway are not understood fully, but may reveal distinctions that will make these oft-parasitic organisms yield their foothold in the vertebrate host.

Abstract PR02: The diverse roles of long noncoding RNAs in the p53 tumor suppressor pathway

The p53 tumor suppressor is a transcription factor, which plays a central role in the cellular response to DNA damage and oncogenic stress. Using genetic approaches in the mouse as well as antisense technology, we have previously shown that the p53-regulated lncRNA, lincRNA-p21 activates the expression of its neighboring gene, the critical G1/S checkpoint mediator, p21. Mechanistically, we found that lincRNA-p21 localizes at its site of transcription, where it acts in cis to stabilize the binding of p53 and its transcriptional co-activator hnRNP-K at the promoter of p21. Importantly, using CRISPR-based approaches to modify the endogenous lincRNA-p21 locus in primary human fibroblasts, we show that the role of lincRNA-p21 in promoting p21 levels is conserved between mouse and human. To expand our understanding of the contribution of lncRNAs to the p53 tumor suppressor pathway, we have performed p53 ChIPseq and RNAseq under conditions of oncogenic stress and identified multiple previously uncharacterized lncRNAs that are regulated in a p53-dependent manner. One of them, lincRNA-Dhx15 is a novel lncRNA expressed from a bidirectional promoter shared with the RNA helicase Dhx15. LincRNA-Dhx15 is a direct target of p53 by virtue of multiple p53 response elements located in its first intron and appears to be preferentially expressed under conditions of senescence. We have generated a conditional knockout of lincRNA-Dhx15 in the mouse and found that absence of lincRNA-Dhx15 leads to impaired induction of senescence following prolonged exposure to DNA damage. Moreover, by gene expression profiling, we observed a downregulation of a subset of p53 target genes, suggesting that lincRNA-Dhx15 plays a global role in the p53 pathway. Surprisingly, we found that lincRNA-Dhx15 is exported to the cytoplasm and appears to promote the translational of p53 itself. The mechanistic basis for lincRNA-Dhx15 function is under active investigation. Altogether, these studies reveal that p53-regulated lncRNAs act through diverse mechanisms to add important layers of regulation to the p53 transcriptional network. Citation Format: Jordan Bendor, Jesse Zamudio, Tyler Jacks, Nadva Dimitrova. The diverse roles of long noncoding RNAs in the p53 tumor suppressor pathway. [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 PR02.