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Effect of the intrinsically disordered region (IDR) within the CLK2 (T343A) mutant in the regulation of intron retention

ABSTRACT: We established the HeLa cells expressing the GFP (control) or CLK2 (T343A) with deletion of the IDR [T343A (dIDR)]. These cells were subjected to RNA-seq for the alternative splicing analysis.

ORGANISM(S): Homo sapiens

PROVIDER: GSE247419 | GEO | 2023/11/09

REPOSITORIES: GEO

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Effect of the intrinsically disordered region (IDR) within the CLK2 (T343A) mutant in the regulation of intron retention

Project description:Effect of the intrinsically disordered region (IDR) within the CLK2 (T343A) mutant in the regulation of intron retention

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Effect of CLK2 expression in the regulation of intron retention during heat shock [RNA-seq]

Project description:Intron retention (IR) constitutes a less explored form of alternative splicing, wherein introns are retained within mature mRNA transcripts. Our investigation demonstrates that the CDC-like kinase 2 (CLK2) undergoes liquid-liquid phase separation (LLPS) within nuclear speckles in response to heat shock (HS). The formation of CLK2 condensates depends on the intrinsically disordered region (IDR) located within the N-terminal amino acids 1-148. Phosphorylation at residue T343 sustains CLK2 kinase activity and facilitates autophosphorylation, thus inhibiting the LLPS activity of the IDR. These CLK2 condensates initiate the reorganization of nuclear speckles, transforming them into larger, rounded structures. Moreover, these condensates facilitate the recruitment of splicing factors into these compartments, potentially restricting their access to mRNA for intron splicing. Consequently, the formation of CLK2 condensates promotes the IR.

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Effect of CLK2 expression in the regulation of intron retention during heat shock

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Replication licensing regulated by an intrinsically disordered region of origin recognition complex

Project description:In eukaryotes, the hetero-hexameric origin recognition complex (ORC) is responsible for assembling Mcm2-7 complex into a head-to-head double hexamer (DH), forming pre-replicative complex (pre-RC) that licenses origin DNA for replication initiation. This process is tightly controlled throughout the cell cycle to ensure accurate duplication of the genome. Here we show that the N-terminal intrinsically disordered region (IDR) of the yeast Orc2 subunit plays a critical role in promoting pre-RC assembly. We found that removing a short segment (residues 175-200) from Orc2-IDR or mutating a key isoleucine (194) in this region significantly inhibits replication initiation across the genome and causes cell death. Although the Orc2-IDR mutants can still assemble the ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) intermediate on DNA, the assembled mutant OCCM exhibits impaired ATP hydrolysis, preventing its conversion into Mcm2-7-ORC (MO) complex and subsequent DH formation. Interestingly, our in vitro assays showed that adding the Orc2-IDR peptide in the pre-RC reactions can rescue this defect. Furthermore, phosphorylation of this Orc2-IDR motif by S-cyclin dependent kinase (S-CDK) blocks its binding to Mcm2, leading to defective pre-RC assembly. Our findings provide crucial mechanistic insights into the multifaceted roles of ORC in modulating MCM loading to support origin licensing during the G1 phase and its regulation to restrict origin firing within the S phase.

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Proteins that interact with CLK2 (WT) or CLK2 (T343A)

Project description:Our results show that CLK2 undergoes liquid-liquid phase separation (LLPS) in response to heat shock stress. Phosphorylation of CLK2 at T343 prevents the LLPS of CLK2. To identify the proteins that recruited to the CLK2 condensates, we immunoprecipitated CLK2 (WT) or (T343A) proteins with FLAG antibody and performed mass spectrometry to detect the interacting proteins.

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An Intrinsically Disordered Region Controlling Condensation of a Circadian Clock Component and Rhythmic Transcription in the Liver [ChIP-seq]

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Project description:RIF1 is a multifunctional protein that plays key roles in the regulation of DNA processing. During repair of DNA double-strand breaks (DSBs), RIF1 functions in the 53BP1-Shieldin pathway that inhibits resection of DNA ends to modulate the cellular decision on which repair pathway to engage. Under conditions of replication stress, RIF1 protects nascent DNA at stalled replication forks from degradation by the DNA2 nuclease. How these RIF1 activities are regulated at the post-translational level has not yet been elucidated. Here, we identified a cluster of conserved ATM/ATR consensus SQ motifs within the intrinsically disordered region (IDR) of mouse RIF1 that are phosphorylated in proliferating B lymphocytes. We found that phosphorylation of the conserved IDR SQ cluster is dispensable for the inhibition of DSB resection by RIF1, but is essential to counteract DNA2-dependent degradation of nascent DNA at stalled replication forks. Therefore, our study identifies a key molecular feature that enables the genome-protective function of RIF1 during DNA replication stress.

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