Genome-wide identification of DNA-PKcs-associated RNAs by RIP-Seq

Song, Zhiquan; Xie, Yufeng; Guo, Zongpei; Yang, Han; Guan, Hua; Liu, Xiaodan; Ma, Teng; Zhou, Ping‐Kun

doi:10.1038/s41392-019-0057-6

Cited by 17 publications

(9 citation statements)

References 8 publications

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“…DNA-PK is well known for its role as a DNA repair complex that is responsible for the repair of DNA double-strand breaks through non-homologous end joining (NHEJ) and maintaining DNA integrity ( 48 ). Moreover, the role of DNA-PK in cells is highly diverse as it can also act as an RNA-dependent protein kinase to phosphorylate hnRNP molecules ( 49 , 50 ) and a recent report indicates that DNA-PK might be involved in alternative splicing of mRNAs ( 51 ). These functions could be involved in the LUCAT1-dependent splicing events that we report in this study.…”

Section: Discussionmentioning

confidence: 99%

The lncRNA LUCAT1 is elevated in inflammatory disease and restrains inflammation by regulating the splicing and stability of NR4A2

Vierbuchen

Agarwal

Johnson

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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The nuclear long non-coding RNA LUCAT1 has previously been identified as a negative feedback regulator of type I interferon and inflammatory cytokine expression in human myeloid cells. Here, we define the mechanistic basis for the suppression of inflammatory gene expression by LUCAT1. Using comprehensive identification of RNA-binding proteins by mass spectrometry as well as RNA immunoprecipitation, we identified proteins important in processing and alternative splicing of mRNAs as LUCAT1-binding proteins. These included heterogeneous nuclear ribonucleoprotein C, M, and A2B1. Consistent with this finding, cells lacking LUCAT1 have altered splicing of selected immune genes. In particular, upon lipopolysaccharide stimulation, the splicing of the nuclear receptor 4A2 (NR4A2) gene was particularly affected. As a consequence, expression of NR4A2 was reduced and delayed in cells lacking LUCAT1. NR4A2-deficient cells had elevated expression of immune genes. These observations suggest that LUCAT1 is induced to control the splicing and stability of NR4A2, which is in part responsible for the anti-inflammatory effect of LUCAT1. Furthermore, we analyzed a large cohort of patients with inflammatory bowel disease as well as asthma and chronic obstructive pulmonary disease. In these patients, LUCAT1 levels were elevated and in both diseases, positively correlated with disease severity. Collectively, these studies define a key molecular mechanism of LUCAT1-dependent immune regulation through post-transcriptional regulation of mRNAs highlighting its role in the regulation of inflammatory disease.

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Section: Discussionmentioning

confidence: 99%

The lncRNA LUCAT1 is elevated in inflammatory disease and restrains inflammation by regulating the splicing and stability of NR4A2

Vierbuchen

Agarwal

Johnson

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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“…For high-throughput sequencing, the cDNA libraries were applied to the Illumina Hiseq2000 system for 150 nt paired-end sequencing by ABLife company. The RIP-seq data analysis was performed as described [ 99 ]. Two replicates were performed for both the RNA-seq and RIP-seq assays.…”

Section: Methodsmentioning

confidence: 99%

The rice blast fungus SR protein 1 regulates alternative splicing with unique mechanisms

et al. 2022

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Serine/arginine-rich (SR) proteins are well known as splicing factors in humans, model animals and plants. However, they are largely unknown in regulating pre-mRNA splicing of filamentous fungi. Here we report that the SR protein MoSrp1 enhances and suppresses alternative splicing in a model fungal plant pathogen Magnaporthe oryzae. Deletion of MoSRP1 caused multiple defects, including reduced virulence and thousands of aberrant alternative splicing events in mycelia, most of which were suppressed or enhanced intron splicing. A GUAG consensus bound by MoSrp1 was identified in more than 94% of the intron or/and proximate exons having the aberrant splicing. The dual functions of regulating alternative splicing of MoSrp1 were exemplified in enhancing and suppressing the consensus-mediated efficient splicing of the introns in MoATF1 and MoMTP1, respectively, which both were important for mycelial growth, conidiation, and virulence. Interestingly, MoSrp1 had a conserved sumoylation site that was essential to nuclear localization and enhancing GUAG binding. Further, we showed that MoSrp1 interacted with a splicing factor and two components of the exon-joining complex via its N-terminal RNA recognition domain, which was required to regulate mycelial growth, development and virulence. In contrast, the C-terminus was important only for virulence and stress responses but not for mycelial growth and development. In addition, only orthologues from Pezizomycotina species could completely rescue defects of the deletion mutants. This study reveals that the fungal conserved SR protein Srp1 regulates alternative splicing in a unique manner.

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“…Alternative splicing is an important mechanism for regulating gene expression and generating proteomic diversity (22). In a previous study, we have shown that the inactivation of DNA-PK activity regulates CD44 alternative splicing (19). In the long-read data, we attempted to gain an alternative splicing landscape in DNA-PK inactivation cells.…”

Section: Transcriptome Structure Analysis and Alternative Splicing Pr...mentioning

confidence: 99%

“…Although the relationship between DNA-PK and the RNA polymerase II complex has been well established, the complex transcriptome dependent on DNA-PK is unknown and much remains to be deciphered about the cellular consequences of DNA-PK. In the past, short-read sequencing-based technology may not have fully resolved the transcriptome landscape dependent on DNA-PK such as alternative splicing ( 19 ), alternative transcription initiation, or alternative transcription termination sites and may further hamper the understanding of DNA-PK functions. Long-read sequencing can directly obtain the full-length transcripts, define novel transcribed regions, and discriminate highly similar isoforms of annotated genes.…”

Section: Introductionmentioning

confidence: 99%

Long-Read Sequencing Annotation of the Transcriptome in DNA-PK Inactivated Cells

Song

Jin

et al. 2022

Front. Oncol.

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The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) with a Ku70/Ku80 heterodimer constitutes the intact DNA-PK kinase, which is an upstream component of the DNA repair machinery that signals the DNA damage, orchestrates the DNA repair, and serves to maintain genome integrity. Beyond its role in DNA damage repair, the DNA-PK kinase is also implicated in transcriptional regulation and RNA metabolism, with an illuminated impact on tumor progression and therapeutic responses. However, the efforts to identify DNA-PK regulated transcriptomes are limited by short-read sequencing to resolve the full complexity of the transcriptome. Therefore, we leveraged the PacBio Single Molecule, Real-Time (SMRT) Sequencing platform to study the transcriptome after DNA-PK inactivation to further underscore the importance of its role in diseases. Our analysis revealed additional novel transcriptome and complex gene structures in the DNA-PK inactivated cells, identifying 8,355 high-confidence new isoforms from 3,197 annotated genes and 523 novel genes. Among them, 380 lncRNAs were identified. We validated these findings using computational approaches and confirmatory transcript quantification with short-read sequencing. Several novel isoforms representing distinct splicing events have been validated through PCR experiments. Our analyses provide novel insights into DNA-PK function in transcriptome regulation and RNA metabolism.

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Genome-wide identification of DNA-PKcs-associated RNAs by RIP-Seq

Cited by 17 publications

References 8 publications

The lncRNA LUCAT1 is elevated in inflammatory disease and restrains inflammation by regulating the splicing and stability of NR4A2

The lncRNA LUCAT1 is elevated in inflammatory disease and restrains inflammation by regulating the splicing and stability of NR4A2

The rice blast fungus SR protein 1 regulates alternative splicing with unique mechanisms

Long-Read Sequencing Annotation of the Transcriptome in DNA-PK Inactivated Cells

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