Enhancement of NEIL1 Protein-initiated Oxidized DNA Base Excision Repair by Heterogeneous Nuclear Ribonucleoprotein U (hnRNP-U) via Direct Interaction

Hegde, Muralidhar L.; Banerjee, Sneha; Hegde, Pavana M.; Bellot, Larry J.; Hazra, Tapas K.; Boldogh, István; Mitra, Sankar

doi:10.1074/jbc.m112.384032

Cited by 53 publications

(84 citation statements)

References 51 publications

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“…The hnRNP U protein has been implicated in many levels of gene regulation, including the maintenance of intranuclear chromosome conformation, and in transcription, RNA splicing, and DNA repair (19,21,46,47). The generation of the Hnrnpu conditional deletion allele provided the opportunity to study the function of hnRNP U in vivo, in specific tissues.…”

Section: Discussionmentioning

confidence: 99%

hnRNP U protein is required for normal pre-mRNA splicing and postnatal heart development and function

Beetz

O’Keeffe

et al. 2015

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

112

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We report that mice lacking the heterogeneous nuclear ribonucleoprotein U (hnRNP U) in the heart develop lethal dilated cardiomyopathy and display numerous defects in cardiac pre-mRNA splicing. Mutant hearts have disorganized cardiomyocytes, impaired contractility, and abnormal excitation-contraction coupling activities. RNA-seq analyses of Hnrnpu mutant hearts revealed extensive defects in alternative splicing of pre-mRNAs encoding proteins known to be critical for normal heart development and function, including Titin and calcium/calmodulin-dependent protein kinase II delta (Camk2d). Loss of hnRNP U expression in cardiomyocytes also leads to aberrant splicing of the pre-mRNA encoding the excitation-contraction coupling component Junctin. We found that the protein product of an alternatively spliced Junctin isoform is N-glycosylated at a specific asparagine site that is required for interactions with specific protein partners. Our findings provide conclusive evidence for the essential role of hnRNP U in heart development and function and in the regulation of alternative splicing.T he expression of more than 95% of human genes is affected by alternative pre-mRNA splicing (AS) (1, 2). Differentially spliced isoforms play distinct roles in a temporally and spatially specific manner (3), and mutations that lead to aberrant splicing are the cause of many human genetic diseases (4). RNA-binding proteins (RBPs) play a central role in the regulation of alternative splicing during development and disease. They function primarily by positively or negatively regulating splice-site recognition by the spliceosome (1). Many RBPs are expressed in specific tissues, and AS is regulated by the combinatorial activities of these factors on specific pre-mRNAs through their interactions with distinct regulatory sequences in premRNA that function as splicing enhancers or silencers (5).The developing heart is one of the best studied systems where splicing changes occur during normal development, and mutations affecting specific splicing outcomes contribute to cardiomyopathy (6, 7). Although these mutations can either disrupt splicing elements or affect the expression of specific splicing factors, the latter mechanism is clearly responsible for the distinct splicing profiles at different developmental stages. For example, the dynamics of alternative splicing during postnatal heart development correlate with expression changes of many RBPs, including CUG-BP, Elavlike family member 1 (CELF1), Muscleblind-like 1 (MBNL1), and FOX proteins (8). Detailed biochemical studies have elucidated the mechanisms by which these splicing factors regulate splicing in a position-and context-dependent manner (9, 10). The function of other RBPs during heart development has also been studied. For example, two of the muscle-specific splicing factors, RBM20 and RBM24, play distinct roles in splicing regulation. RBM20 mainly acts as a splicing repressor, as its absence leads to multiple exon inclusion events in the heart. For example, the Titin gene is one of...

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Section: Discussionmentioning

confidence: 99%

hnRNP U protein is required for normal pre-mRNA splicing and postnatal heart development and function

Beetz

O’Keeffe

et al. 2015

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

112

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“…Similar repair reactions were carried with a circular duplex DNA substrate containing a single 5-OHU residue that was generated as described previously (18,34). Briefly, the plasmid pUC19CPD containing two single-strand nicking sites on the same strand, 32 nucleotides apart, was digested with N.BstNB1 (New England Biolabs) and heated at 65°C for 10 min to dissociate the 32-mer oligonucleotide (5Ј-GCG GAT ATT AAT GTG ACG GTA GCG AGT CGC TC-3Ј) that was removed by annealing with an of excess biotinylated complementary 32-nucleotide oligonucleotide followed by binding to streptavidinagarose Dynabeads (Sigma).…”

Section: Methodsmentioning

confidence: 99%

The Interaction between Polynucleotide Kinase Phosphatase and the DNA Repair Protein XRCC1 Is Critical for Repair of DNA Alkylation Damage and Stable Association at DNA Damage Sites

Della-Maria

Hegde

McNeill

et al. 2012

Journal of Biological Chemistry

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Background: XRCC1 interacts with multiple DNA repair proteins. Results: Identification of mutant versions of XRCC1 that are defective in binding with a different single partner. Conclusion:Interaction between XRCC1 and polynucleotide kinase 3Ј-phosphatase is critical for the retention of XRCC1 at DNA damage sites and DNA damage repair. Significance: Insights into function of one of three DNA end processing factors that bind to the same region of XRCC1.

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“…LNK-92101-KI01; OLink Bioscience) per the manufacturer's instructions. The nuclei were counterstained with DAPI, and the PLA signals visualized in a fluorescence microscope (Olympus) at 200× magnification (56,64).…”

Section: Methodsmentioning

confidence: 99%

“…The 32 P-5′-end labeling of the oligos, annealing, and purification of labeled oligos were described previously (11). Circular plasmid substrate, pUC19CPD containing a single oxidized base lesion, 5-OHU, was generated as described previously (32,56,57). Covalently closed form I plasmid was purified by CsCl centrifugation, and the presence of the 5-OHU lesion was verified by agarose gel electrophoresis after treatment with NEIL1, which converted the plasmid to a nicked circle (form II).…”

Section: Methodsmentioning

confidence: 99%

Prereplicative repair of oxidized bases in the human genome is mediated by NEIL1 DNA glycosylase together with replication proteins

Hegde

Hegde²,

Bellot³

et al. 2013

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

Self Cite

131

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Base oxidation by endogenous and environmentally induced reactive oxygen species preferentially occurs in replicating single-stranded templates in mammalian genomes, warranting prereplicative repair of the mutagenic base lesions. It is not clear how such lesions (which, unlike bulky adducts, do not block replication) are recognized for repair. Furthermore, strand breaks caused by base excision from ssDNA by DNA glycosylases, including Nei-like (NEIL) 1, would generate double-strand breaks during replication, which are not experimentally observed. NEIL1, whose deficiency causes a mutator phenotype and is activated during the S phase, is present in the DNA replication complex isolated from human cells, with enhanced association with DNA in S-phase cells and colocalization with replication foci containing DNA replication proteins. Furthermore, NEIL1 binds to 5-hydroxyuracil, the oxidative deamination product of C, in replication protein A-coated ssDNA template and inhibits DNA synthesis by DNA polymerase δ. We postulate that, upon encountering an oxidized base during replication, NEIL1 initiates prereplicative repair by acting as a "cowcatcher" and preventing nascent chain growth. Regression of the stalled replication fork, possibly mediated by annealing helicases, then allows lesion repair in the reannealed duplex. This model is supported by our observations that NEIL1, whose deficiency slows nascent chain growth in oxidatively stressed cells, is stimulated by replication proteins in vitro. Furthermore, deficiency of the closely related NEIL2 alone does not affect chain elongation, but combined NEIL1/2 deficiency further inhibits DNA replication. These results support a mechanism of NEIL1-mediated prereplicative repair of oxidized bases in the replicating strand, with NEIL2 providing a backup function.genome damage repair | replication fork stalling | oxidized base repair at DNA replication fork S everal dozen oxidatively modified, and mostly mutagenic, bases are induced in the genomes of aerobic organisms by endogenous and environmentally induced reactive oxygen species (ROS) (1, 2). For example, 5-hydroxyuracil (5-OHU), a predominant lesion generated by oxidative deamination of C, is mutagenic because of its mispairing with A (3). The bases in the single-stranded (ss) replicating DNA template are particularly prone to oxidation (4); the lack of their repair before replication could fix the mutations. The bulky base adducts if formed in the template strand would block replication and trigger DNA damage-response signaling. In contrast, oxidized bases with minor modifications, which are continuously formed in much higher abundance than the bulky adducts, would mostly allow replication. This raises the question of how these bases are marked for repair before replication to avoid mutagenic consequences. Oxidized base repair in mammalian genomes occurs primarily via the base excision repair (BER) pathway which is initiated with lesion base excision mediated by one of five major DNA glycosylases belonging to the Nth or...

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Enhancement of NEIL1 Protein-initiated Oxidized DNA Base Excision Repair by Heterogeneous Nuclear Ribonucleoprotein U (hnRNP-U) via Direct Interaction

Cited by 53 publications

References 51 publications

hnRNP U protein is required for normal pre-mRNA splicing and postnatal heart development and function

hnRNP U protein is required for normal pre-mRNA splicing and postnatal heart development and function

The Interaction between Polynucleotide Kinase Phosphatase and the DNA Repair Protein XRCC1 Is Critical for Repair of DNA Alkylation Damage and Stable Association at DNA Damage Sites

Prereplicative repair of oxidized bases in the human genome is mediated by NEIL1 DNA glycosylase together with replication proteins

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