bims-rimeca 2020-12-06 papers

bims-rimeca

Biomed News

on RNA methylation in cancer

Issue of 2020‒12‒06
nine papers selected by
Sk Ramiz Islam
Saha Institute of Nuclear Physics

Programmable N6-methyladenosine modification of CDCP1 mRNA by RCas9-methyltransferase like 3 conjugates promotes bladder cancer development.
CIGAR-seq, a CRISPR/Cas-based method for unbiased screening of novel mRNA modification regulators.
XG-ac4C: identification of N4-acetylcytidine (ac4C) in mRNA using eXtreme gradient boosting with electron-ion interaction pseudopotentials.
Predicting sites of epitranscriptome modifications using unsupervised representation learning based on generative adversarial networks.
The impact of epitranscriptomic marks on post-transcriptional regulation in plants.
Longitudinal epitranscriptome profiling reveals the crucial role of N6-methyladenosine methylation in porcine prenatal skeletal muscle development.
Epitranscriptomic m6A regulation following spinal cord injury.
Transcriptome-Wide m6A Methylation in Skin Lesions From Patients With Psoriasis Vulgaris.
KIAA1429 promotes the progression of lung adenocarcinoma by regulating the m6A level of MUC3A.

Mol Cancer. 2020 Dec 03. 19(1): 169

Programmable N6-methyladenosine modification of CDCP1 mRNA by RCas9-methyltransferase like 3 conjugates promotes bladder cancer development.

Xiaoling Ying, Xu Jiang, Haiqing Zhang, Bixia Liu, Yapeng Huang, Xiaowei Zhu, Defeng Qi, Gang Yuan, Junhang Luo, Weidong Ji.

Accumulating evidence has revealed significant roles for N6-methyladenosine (m 6 A) modification in the development of various cancers. We previously demonstrated an oncogenic role of m 6 A-modified CUB domain containing protein 1 (CDCP1) in bladder cancer (BC) progression. However, the biological functions and underlying molecular mechanisms of engineered programmable m 6 A modification of CDCP1 mRNA in BC remain obscure. Here, we established a targeted m 6 A RNA methylation system by fusing the catalytic domain of methyltransferase like 3 (METTL3CD) to RCas9 as the RNA-targeting module. The constructed RCas9- METTL3 retained methylation activity and mediated efficient site-specific m 6 A installation in the presence of a cognate single guide RNA and short protospacer adjacent motif-containing ssDNA molecule . Subsequently, targeting m 6 A installation onto the 3' untranslated region of CDCP1 promoted CDCP1 mRNA translation and facilitated BC development in vitro and in vivo. Our findings demonstrate that the RCas9-METTL3 system mediates efficient sitespecific m 6 A installation on CDCP1 mRNA and promotes BC development. Thus, the RCas9-METTL3 system provides a new tool for studying m 6 A function and a potential strategy for BC epitranscriptome-modulating therapies.

DOI: https://doi.org/10.1186/s12943-020-01289-0
Mol Syst Biol. 2020 Nov;16(11): e10025

CIGAR-seq, a CRISPR/Cas-based method for unbiased screening of novel mRNA modification regulators.

Liang Fang, Wen Wang, Guipeng Li, Li Zhang, Jun Li, Diwen Gan, Jiao Yang, Yisen Tang, Zewen Ding, Min Zhang, Wenhao Zhang, Daqi Deng, Zhengyu Song, Qionghua Zhu, Huanhuan Cui, Yuhui Hu, Wei Chen.

  Cellular RNA is decorated with over 170 types of chemical modifications. Many modifications in mRNA, including m6 A and m5 C, have been associated with critical cellular functions under physiological and/or pathological conditions. To understand the biological functions of these modifications, it is vital to identify the regulators that modulate the modification rate. However, a high-throughput method for unbiased screening of these regulators is so far lacking. Here, we report such a method combining pooled CRISPR screen and reporters with RNA modification readout, termed CRISPR integrated gRNA and reporter sequencing (CIGAR-seq). Using CIGAR-seq, we discovered NSUN6 as a novel mRNA m5 C methyltransferase. Subsequent mRNA bisulfite sequencing in HAP1 cells without or with NSUN6 and/or NSUN2 knockout showed that NSUN6 and NSUN2 worked on non-overlapping subsets of mRNA m5 C sites and together contributed to almost all the m5 C modification in mRNA. Finally, using m1 A as an example, we demonstrated that CIGAR-seq can be easily adapted for identifying regulators of other mRNA modification.

Keywords:  CIGAR-seq; NSUN6; m5C modification; mRNA modification; pooled CRISPR screen

DOI:  https://doi.org/10.15252/msb.202010025
Sci Rep. 2020 Dec 01. 10(1): 20942

XG-ac4C: identification of N4-acetylcytidine (ac4C) in mRNA using eXtreme gradient boosting with electron-ion interaction pseudopotentials.

Waleed Alam, Hilal Tayara, Kil To Chong.

N4-acetylcytidine (ac4C) is a post-transcriptional modification in mRNA which plays a major role in the stability and regulation of mRNA translation. The working mechanism of ac4C modification in mRNA is still unclear and traditional laboratory experiments are time-consuming and expensive. Therefore, we propose an XG-ac4C machine learning model based on the eXtreme Gradient Boost classifier for the identification of ac4C sites. The XG-ac4C model uses a combination of electron-ion interaction pseudopotentials and electron-ion interaction pseudopotentials of trinucleotide of the nucleotides in ac4C sites. Moreover, Shapley additive explanations and local interpretable model-agnostic explanations are applied to understand the importance of features and their contribution to the final prediction outcome. The obtained results demonstrate that XG-ac4C outperforms existing state-of-the-art methods. In more detail, the proposed model improves the area under the precision-recall curve by 9.4% and 9.6% in cross-validation and independent tests, respectively. Finally, a user-friendly web server based on the proposed model for ac4C site identification is made freely available at http://nsclbio.jbnu.ac.kr/tools/xgac4c/ .

DOI: https://doi.org/10.1038/s41598-020-77824-2
Front Phys. 2020 Jun;pii: 196. [Epub ahead of print]8

Predicting sites of epitranscriptome modifications using unsupervised representation learning based on generative adversarial networks.

Sirajul Salekin, Milad Mostavi, Yu-Chiao Chiu, Yidong Chen, Jianqiu Michelle Zhang, Yufei Huang.

  Epitranscriptome is an exciting area that studies different types of modifications in transcripts and the prediction of such modification sites from the transcript sequence is of significant interest. However, the scarcity of positive sites for most modifications imposes critical challenges for training robust algorithms. To circumvent this problem, we propose MR-GAN, a generative adversarial network (GAN) based model, which is trained in an unsupervised fashion on the entire pre-mRNA sequences to learn a low dimensional embedding of transcriptomic sequences. MR-GAN was then applied to extract embeddings of the sequences in a training dataset we created for eight epitranscriptome modifications, including m6A, m1A, m1G, m2G, m5C, m5U, 2'-O-Me, Pseudouridine (Ψ) and Dihydrouridine (D), of which the positive samples are very limited. Prediction models were trained based on the embeddings extracted by MR-GAN. We compared the prediction performance with the one-hot encoding of the training sequences and SRAMP, a state-of-the-art m6A site prediction algorithm and demonstrated that the learned embeddings outperform one-hot encoding by a significant margin for up to 15% improvement. Using MR-GAN, we also investigated the sequence motifs for each modification type and uncovered known motifs as well as new motifs not possible with sequences directly. The results demonstrated that transcriptome features extracted using unsupervised learning could lead to high precision for predicting multiple types of epitranscriptome modifications, even when the data size is small and extremely imbalanced.

Keywords:  N6-methyladenosine (m6A); RNA modification site prediction; epitranscriptome; generative adversarial networks (GAN); methylated RNA immunoprecipitation sequencing (MeRIP-Seq); unsupervised representation learning

DOI:  https://doi.org/10.3389/fphy.2020.00196
Brief Funct Genomics. 2020 Dec 04. pii: elaa021. [Epub ahead of print]

The impact of epitranscriptomic marks on post-transcriptional regulation in plants.

Xiang Yu, Bishwas Sharma, Brian D Gregory.

  Ribonucleotides within the various RNA molecules in eukaryotes are marked with more than 160 distinct covalent chemical modifications. These modifications include those that occur internally in messenger RNA (mRNA) molecules such as N6-methyladenosine (m6A) and 5-methylcytosine (m5C), as well as those that occur at the ends of the modified RNAs like the non-canonical 5' end nicotinamide adenine dinucleotide (NAD+) cap modification of specific mRNAs. Recent findings have revealed that covalent RNA modifications can impact the secondary structure, translatability, functionality, stability and degradation of the RNA molecules in which they are included. Many of these covalent RNA additions have also been found to be dynamically added and removed through writer and eraser complexes, respectively, providing a new layer of epitranscriptome-mediated post-transcriptional regulation that regulates RNA quality and quantity in eukaryotic transcriptomes. Thus, it is not surprising that the regulation of RNA fate mediated by these epitranscriptomic marks has been demonstrated to have widespread effects on plant development and the responses of these organisms to abiotic and biotic stresses. In this review, we highlight recent progress focused on the study of the dynamic nature of these epitranscriptome marks and their roles in post-transcriptional regulation during plant development and response to environmental cues, with an emphasis on the mRNA modifications of non-canonical 5' end NAD+ capping, m6A and several other internal RNA modifications.

Keywords:  NAD+ capping; Post-transcriptional regulation; RNA methylation; RNA modifications; RNA stability; epitranscriptome

DOI:  https://doi.org/10.1093/bfgp/elaa021
J Genet Genomics. 2020 Sep 07. pii: S1673-8527(20)30131-4. [Epub ahead of print]

Longitudinal epitranscriptome profiling reveals the crucial role of N6-methyladenosine methylation in porcine prenatal skeletal muscle development.

Xinxin Zhang, Yilong Yao, Jinghua Han, Yalan Yang, Yun Chen, Zhonglin Tang, Fei Gao.

  N6-methyladenosine (m6A) represents the most abundantly occurring mRNA modification and is involved in the regulation of skeletal muscle development. However, the status and function of m6A methylation in prenatal myogenesis remains unclear. In this study, we first demonstrated that knockdown of METTL14, an m6A methyltransferase, inhibited the differentiation and promoted the proliferation of C2C12 myoblast cells. Then, using a refined m6A-specific methylated RNA immunoprecipitation [RIP] with nextgeneration sequencing (MeRIP-seq) method that is optimal for use with samples containing small amounts of RNA, we performed transcriptome-wide m6A profiling for six prenatal skeletal muscle developmental stages spanning two important waves of porcine myogenesis. The results revealed that, along with a continuous decrease in the mRNA expression of the m6A reader protein insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), the m6A methylome underwent highly dynamic changes across different development stages, with most of the affected genes being enriched in pathways related to skeletal muscle development. RNA immunoprecipitation confirmed that IGF2BP1 targets 76 genes involved in pathways associated with muscle development, including the key marker genes MYH2 and MyoG. Moreover, small interfering RNA (siRNA)-mediated knockdown of IGF2BP1 induced phenotypic changes in C2C12 myoblasts similar to those observed with knockdown of METTL14. In conclusion, we clarified the dynamics of m6A methylation and identified key genes involved in the regulatory network of porcine skeletal muscle development.

Keywords:  IGF2BP1; Myoblast; N(6)-methyladenosine; Skeletal muscle

DOI:  https://doi.org/10.1016/j.jgg.2020.07.003
J Neurosci Res. 2020 Dec 03.

Epitranscriptomic m6A regulation following spinal cord injury.

Lingyan Xing, Yunyun Cai, Tuo Yang, Weiwei Yu, Mengdie Gao, Rui Chai, Sujun Ding, Jinhuan Wei, Jingying Pan, Gang Chen.

  RNA methylation is involved in multiple physiological and pathological processes. However, the role of RNA methylation in spinal cord regeneration has not been reported. In this study, we find an altered m6A (N6-methyladenosine) RNA methylation profiling following zebrafish spinal cord injury (SCI), in line with an altered transcription level of the m6A methylase Mettl3. Interestingly, many of the differential m6A-tagged genes associated with neural regeneration are hypomethylated, but their transcription levels are upregulated in SCI. Moreover, we find that METTL3 may be important for spinal cord regeneration. We also show a conserved feature of METTL3 changes in mouse SCI model, in which the expression of METTL3 is increased in both astrocytes and neural stem cells. Together, our results indicate that m6A RNA methylation is dynamic and conserved following SCI and may contribute to spinal cord regeneration.

Keywords:  Mettl3 (Methyltransferase Like 3); RNA methylation; RRID:AB_11212597; RRID:AB_2338072; RRID:AB_2338840; RRID:AB_2721254; RRID:AB_305313; m6A (N6-methyladenosine); spinal cord injury

DOI:  https://doi.org/10.1002/jnr.24763
Front Cell Dev Biol. 2020 ;8 591629

Transcriptome-Wide m6A Methylation in Skin Lesions From Patients With Psoriasis Vulgaris.

Ya-Nan Wang, Hong-Zhong Jin.

  N6-methyladenosine (m6A) methylation, as the most prevalent internal RNA modification, has been revealed to play critical roles in various biological functions. In this study, we performed m6A transcriptome-wide profiling in three kinds of skin tissue: involved psoriatic skin (PP), uninvolved psoriatic skin (PN), and healthy control skin samples (NN). The findings revealed that transcripts of PP contained the fewest m6A peaks and lowest m6A peak density. The greatest differences of m6A methylation were observed in the PP vs. NN and PP vs. PN comparisons. Intriguingly, in these comparisons, hypermethylated m6A was mainly enriched within the CDSs and 3'UTRs, while hypomethylated m6A was not only enriched within CDSs and 3'UTRs, but also within 5'UTRs. GO and KEGG pathway analyses indicated that hypermethylated transcripts in PP were particularly associated with response-associated terms, cytokine production, and olfactory transduction. Meanwhile, hypomethylated transcripts in PP were mainly associated with development-related processes and the Wnt signaling pathway. In addition, we discovered that 19.3-48.4% of the differentially expressed transcripts in psoriasis vulgaris were modified by m6A, and that transcripts with lower expression were more preferentially modified by m6A. Moreover, upregulation of gene expression was often accompanied by upregulation of m6A methylation, suggesting a regulatory role of m6A in psoriasis vulgaris gene expression.

Keywords:  MeRIP-seq; N6-methyladenosine; RNA modification; m6A methylation; psoriasis vulgaris

DOI:  https://doi.org/10.3389/fcell.2020.591629
Pathol Res Pract. 2020 Nov 12. pii: S0344-0338(20)32139-7. [Epub ahead of print]217 153284

KIAA1429 promotes the progression of lung adenocarcinoma by regulating the m6A level of MUC3A.

Wenhua Zhao, Yuan Xie.

  OBJECTIVE: Lung adenocarcinoma (LUAD) is one of the most frequently occurring human malignancies worldwide, but its potential molecular mechanism has not yet been fully elucidated. N6-methyladenosine (m6A), the most common internal chemical modification of mRNAs, is implicated in diverse pathological processes in different human malignancies, but its functions in LUAD remain elusive. The current study aimed to investigate the function and molecular mechanism of KIAA1429 in LUAD.METHODS: The KIAA1429 expression level in LUAD tissues was assessed using databases and was detected in LUAD cells and tissues via quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot. m6A levels in LUAD tissues and cells were quantified. Next, correlation between the KIAA1429 expression level and the clinical and pathological features and prognosis of patients with LUAD was analyzed. Further, KIAA1429 levels were decreased, and LUAD cell proliferation, migration, invasion, and cycle were assessed. Prediction websites revealed the aberrant expression and probable methylation modification of MUC3A in LUAD, and correlation between MUC3A and KIAA1429 was analyzed. Ultimately, the impact of the KIAA1429 expression on MUC3A-mediated malignant phenotypes of LUAD was examined by a torsion test.
RESULTS: KIAA1429 expression was remarkably high and m6A level was aberrantly elevated in LUAD cells and tissues. In addition, high KIAA1429 expression indicated a larger tumor diameter, higher tumor-node-metastasis stage, greater proneness to lymph node and distant metastasis, and lower overall survival rate. siRNA-triggered KIAA1429 downregulation dramatically suppressed LUAD cell proliferation, migration, invasion, and cell cycle arrest in the G1 phase. Bioinformatics analysis revealed that MUC3A was expressed in LUAD at an unusually high level and may be methylated under the control of KIAA1429. Western blot, qRT-PCR, and correlation analyses revealed a positive correlation between KIAA1429 expression level and MUC3A. Finally, torsion test results revealed that low KIAA1429 expression reversed LUAD cell migration, proliferation, and invasion facilitated by low MUC3A expression as well as cell cycle arrest in the G1 phase.
CONCLUSION: KIAA1429 exhibited an unusually high expression in LUAD cells and tissues, and high KIAA1429 expression was correlated with the clinical and pathological features of patients with LUAD, thereby leading to an unsatisfactory prognosis. Furthermore, KIAA1429 regulates MUC3A expression through m6A modification to modulate LUAD cells to proliferate, migrate, invade, and induce cell cycle arrest.

Keywords:  Cell proliferation; KIAA1429; Lung adenocarcinoma; MUC3A; Methylation

DOI:  https://doi.org/10.1016/j.prp.2020.153284