RNA cytosine methylation and methyltransferases mediate chromatin organization and 5-azacytidine response and resistance in leukaemia

Cheng, Jian; Chen, Li; Li, Yuan; Cloe, Adam; Ming, Yue; Wei, Jiangbo; Watanabe, Kenneth A.; Shammo, Jamile M.; Anastasi, John; Shen, Qi; Larson, Richard A.; He, Chuan; Beau, Michelle M. Le; Vardiman, James W.

doi:10.1038/s41467-018-03513-4

Cited by 160 publications

(152 citation statements)

References 63 publications

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“…Additionally, other groups reported that low levels of NSUN1 reduce cell growth in leukaemia cells, which is in line with findings that NSUN1 promotes cell proliferation. Moreover, high NSUN1 expression results in increased tumour aggressiveness and augmented 5-azacytidine (5-AZA) resistance in two leukaemia cell lines (Bantis et al, 2004; Cheng et al, 2018; Saijo et al, 2001). Thus, NSUN1 might be considered as an example of ‘antagonistic pleiotropy’.…”

Section: Discussionmentioning

confidence: 99%

The ribosomal RNA m⁵C methyltransferase NSUN-1 modulates healthspan and oogenesis inCaenorhabditis elegans

Heissenberger

Krammer

Rollins

et al. 2020

Preprint

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21Our knowledge about the repertoire of ribosomal RNA modifications and the enzymes 22 responsible for installing them is constantly expanding. Previously, we reported that NSUN-5 23 is responsible for depositing m 5 C at position C2381 on the 26S rRNA in Caenorhabditis 24 elegans. 25 Here, we show that NSUN-1 is writing the second known 26S rRNA m 5 C at position C2982. 26 Depletion of nsun-1 or nsun-5 improved locomotion at midlife and resistance against heat 27 stress, however, only soma-specific knockdown of nsun-1 extended lifespan. Moreover, 28 soma-specific knockdown of nsun-1 reduced body size and impaired fecundity, suggesting 29 non-cell-autonomous effects. While ribosome biogenesis and global protein synthesis were 30 unaffected by nsun-1 depletion, translation of specific mRNAs was remodelled leading to 31 reduced production of collagens, loss of structural integrity of the cuticle and impaired barrier 32 function. 33 We conclude that loss of a single enzyme required for rRNA methylation has profound and 34 highly specific effects on organismal physiology. 102Therefore, we investigated the RNA substrate of NSUN-1 (also formerly known as NOL-1, 103 NOL-2 or W07E6.1) and its potential roles in worm physiology. NSUN-1 is also a member 104 of the NOP2/Sun RNA methyltransferase family. Since there are only two known m 5 C 105 residues on worm 26S rRNA (Sharma and Lafontaine, 2015; Trixl and Lusser, 2019), one of 106 them at C2381, being installed by NSUN-5, we speculated that NSUN-1 might be required 107 6 for introducing the second m 5 C residue at position C2982. Notably, both 26S m 5 C sites are 108 localized close to important functional regions of the ribosome, and they are highly conserved 109 during evolution ( Fig. 1A,B). 110In order to test if NSUN-1 is involved in large ribosomal subunit m 5 C methylation, 26S 111 rRNA was purified from worms treated with siRNAs specific to NSUN-1-encoding mRNAs 112 on sucrose gradients, digested to single nucleosides and analysed by quantitative HPLC. In 113 our HPLC assay, the m 5 C nucleoside eluted at 12 min, as established with a m 5 C calibration 114 control (data not shown). 115The depletion of NSUN-1 was conducted in two genetic backgrounds: N2 (wildtype), and 116 NL2099 (an RNAi-hypersensitive strain due to mutation in rrf-3) (Figure 1-figure 117 supplement 1). Treating N2 worms with an empty control vector, not expressing any RNAi, 118did not significantly reduce the levels of 26S rRNA m 5 C methylation (Fig. 1C, 97% instead 119 of 100%). Interestingly, treating N2 worms with an RNAi construct targeting nsun-1 led to a 120 reduction of 26S rRNA m 5 C methylation by 35% (Fig. 1C). In the NL2099 strain, nsun-1 121 RNAi treatment also led to a reduction of 26S rRNA m 5 C methylation by 26% ( Fig. 1D). 122As there are only two known modified m 5 C residues on worm 26S rRNA, and since one of 123 them is introduced by NSUN-5 (Adamla et al., 2019; Schosserer et al., 2015), a complete loss 124 of NSUN-1 activity was expected to result in a 50% decrease...

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Section: Discussionmentioning

confidence: 99%

The ribosomal RNA m⁵C methyltransferase NSUN-1 modulates healthspan and oogenesis inCaenorhabditis elegans

Heissenberger

Krammer

Rollins

et al. 2020

Preprint

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“…NOP2 promotes HIV-1 latency by competing with Tat for TAR binding binding with BRD4 in 5-AZA-resistant leukemia cell lines [15]. In terms of the relevance to HIV-1 study, an earlier proteomic study identified NOP2 as an RNA binding protein that associates with HIV-1 5'UTR [16].…”

Section: Plos Pathogensmentioning

confidence: 99%

Nucleolar protein NOP2/NSUN1 suppresses HIV-1 transcription and promotes viral latency by competing with Tat for TAR binding and methylation

et al. 2020

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Recent efforts have been paid to identify previously unrecognized HIV-1 latency-promoting genes (LPGs) that can potentially be targeted for eradication of HIV-1 latent reservoirs. From our earlier orthologous RNAi screens of host factors regulating HIV-1 replication, we identified that the nucleolar protein NOP2/NSUN1, a m5C RNA methyltransferase (MTase), is an HIV-1 restriction factor. Loss-and gain-of-function analyses confirmed that NOP2 restricts HIV-1 replication. Depletion of NOP2 promotes the reactivation of latently infected HIV-1 proviruses in multiple cell lines as well as primary CD4 + T cells, alone or in combination with latency-reversing agents (LRAs). Mechanistically, NOP2 associates with HIV-1 5' LTR, interacts with HIV-1 TAR RNA by competing with HIV-1 Tat protein, as well as contributes to TAR m5C methylation. RNA MTase catalytic domain (MTD) of NOP2 mediates its competition with Tat and binding with TAR. Overall, these findings verified that NOP2 suppresses HIV-1 transcription and promotes viral latency.

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“…Several studies have further suggested regulatory roles for m 5 C in mRNAs. For example, it was shown in the context of leukaemia that m 5 C in nascent RNA mediates formation of specific active chromatin structures [67]. m 5 C can also guide systemic mRNA transport in plants [62], promote nuclear export of mammalian mRNA in conjunction with ALYREF [49] and enhance mammalian and zebrafish mRNA stability facilitated by YBX1 [47,51].…”

Section: Introductionmentioning

confidence: 99%

Multiple links between 5-methylcytosine content of mRNA and translation

et al. 2020

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Background: 5-Methylcytosine (m 5 C) is a prevalent base modification in tRNA and rRNA but it also occurs more broadly in the transcriptome, including in mRNA, where it serves incompletely understood molecular functions. In pursuit of potential links of m 5 C with mRNA translation, we performed polysome profiling of human HeLa cell lysates and subjected RNA from resultant fractions to efficient bisulfite conversion followed by RNA sequencing (bsRNA-seq). Bioinformatic filters for rigorous site calling were devised to reduce technical noise.Results: We obtained~1000 candidate m 5 C sites in the wider transcriptome, most of which were found in mRNA. Multiple novel sites were validated by amplicon-specific bsRNA-seq in independent samples of either human HeLa, LNCaP and PrEC cells. Furthermore, RNAi-mediated depletion of either the NSUN2 or TRDMT1 m 5 C:RNA methyltransferases showed a clear dependence on NSUN2 for the majority of tested sites in both mRNAs and noncoding RNAs. Candidate m 5 C sites in mRNAs are enriched in 5′UTRs and near start codons and are embedded in a local context reminiscent of the NSUN2-dependent m 5 C sites found in the variable loop of tRNA. Analysing mRNA sites across the polysome profile revealed that modification levels, at bulk and for many individual sites, were inversely correlated with ribosome association. Conclusions: Our findings emphasise the major role of NSUN2 in placing the m 5 C mark transcriptome-wide. We further present evidence that substantiates a functional interdependence of cytosine methylation level with mRNA translation. Additionally, we identify several compelling candidate sites for future mechanistic analysis.

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RNA cytosine methylation and methyltransferases mediate chromatin organization and 5-azacytidine response and resistance in leukaemia

Cited by 160 publications

References 63 publications

The ribosomal RNA m⁵C methyltransferase NSUN-1 modulates healthspan and oogenesis inCaenorhabditis elegans

The ribosomal RNA m⁵C methyltransferase NSUN-1 modulates healthspan and oogenesis inCaenorhabditis elegans

Nucleolar protein NOP2/NSUN1 suppresses HIV-1 transcription and promotes viral latency by competing with Tat for TAR binding and methylation

Multiple links between 5-methylcytosine content of mRNA and translation

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RNA cytosine methylation and methyltransferases mediate chromatin organization and 5-azacytidine response and resistance in leukaemia

Cited by 160 publications

References 63 publications

The ribosomal RNA m5C methyltransferase NSUN-1 modulates healthspan and oogenesis inCaenorhabditis elegans

The ribosomal RNA m5C methyltransferase NSUN-1 modulates healthspan and oogenesis inCaenorhabditis elegans

Nucleolar protein NOP2/NSUN1 suppresses HIV-1 transcription and promotes viral latency by competing with Tat for TAR binding and methylation

Multiple links between 5-methylcytosine content of mRNA and translation

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The ribosomal RNA m⁵C methyltransferase NSUN-1 modulates healthspan and oogenesis inCaenorhabditis elegans

The ribosomal RNA m⁵C methyltransferase NSUN-1 modulates healthspan and oogenesis inCaenorhabditis elegans