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Initiation of Parental Genome Reprogramming in Fertilized Oocyte by Splicing Kinase SRPK1-Catalyzed Protamine Phosphorylation.

ABSTRACT: The paternal genome undergoes a massive exchange of histone with protamine for compaction into sperm during spermiogenesis. Upon fertilization, this process is potently reversed, which is essential for parental genome reprogramming and subsequent activation; however, it remains poorly understood how this fundamental process is initiated and regulated. Here, we report that the previously characterized splicing kinase SRPK1 initiates this life-beginning event by catalyzing site-specific phosphorylation of protamine, thereby triggering protamine-to-histone exchange in the fertilized oocyte. Interestingly, protamine undergoes a DNA-dependent phase transition to gel-like condensates and SRPK1-mediated phosphorylation likely helps open up such structures to enhance protamine dismissal by nucleoplasmin (NPM2) and enable the recruitment of HIRA for H3.3 deposition. Remarkably, genome-wide assay for transposase-accessible chromatin sequencing (ATAC-seq) analysis reveals that selective chromatin accessibility in both sperm and MII oocytes is largely erased in early pronuclei in a protamine phosphorylation-dependent manner, suggesting that SRPK1-catalyzed phosphorylation initiates a highly synchronized reorganization program in both parental genomes.

SUBMITTER: Gou LT

PROVIDER: S-EPMC7190278 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Initiation of Parental Genome Reprogramming in Fertilized Oocyte by Splicing Kinase SRPK1-Catalyzed Protamine Phosphorylation

Project description:We performed ATAC-seq in mouse sperms and fertilized eggs with wild-type or P1 mutation (phosphorylation sites by SRPK1) to capture early genome reprogramming events ~5 hours post insemination. Surprisingly, we detected broadly distributed ATAC-seq reads across the mouse genome to paternal and maternal gametes formed with wild-type and single mutant sperm, and in contrast, we saw individual peaks with zygotes formed with the double mutant sperm. After assigning to paternal and maternal genomes, we found that the reads assignable to paternal genome was decreased with single and double mutants, compare with wild-type. Furthermore, we found the paternal genome in eggs fertilized with the double mutant sperm essentially retained all sperm-specific ATAC-seq peaks, which were also overlapped with the published H3.3 ChIP-seq signals on wild-type sperm, mostly corresponding to TSSs. Strikingly, the ATAC-seq signals on the maternal genome from eggs fertilized with the double mutant P1 sperm were literally identical to those in MII oocyte. Based on these data, we draw two important conclusions: (i) dramatic chromatin remodeling takes place in a highly coordinated fashion in both paternal and maternal pronuclei before they merge, and (ii) SRPK1-catalyzed protamine phosphorylation initiates such synchronized genome reprogramming in both gametes.

2020-03-16 | GSE136403 | GEO

Initiation of Parental Genome Reprogramming in Fertilized Oocyte by Splicing Kinase SRPK1-Catalyzed Protamine Phosphorylation

Project description:Initiation of Parental Genome Reprogramming in Fertilized Oocyte by Splicing Kinase SRPK1-Catalyzed Protamine Phosphorylation

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Long non-coding RNA MALAT1 increases AKAP-9 expression by promoting SRPK1-catalyzed SRSF1 phosphorylation in colorectal cancer cells.

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Changes in cortical endoplasmic reticulum clusters in the fertilized mouse oocyte†.

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Regiospecific phosphorylation control of the SR protein ASF/SF2 by SRPK1.

Project description:SR proteins (splicing factors containing arginine-serine repeats) are essential factors that control the splicing of precursor mRNA by regulating multiple steps in spliceosome development. The prototypical SR protein ASF/SF2 (human alternative splicing factor) contains two N-terminal RNA recognition motifs (RRMs) (RRM1 and RRM2) and a 50-residue C-terminal RS (arginine-serine-rich) domain that can be phosphorylated at numerous serines by the protein kinase SR-specific protein kinase (SRPK) 1. The RS domain [C-terminal domain that is rich in arginine-serine repeats (residues 198-248)] is further divided into N-terminal [RS1: N-terminal portion of the RS domain (residues 198-227)] and C-terminal [RS2: C-terminal portion of the RS domain (residues 228-248)] segments whose modification guides the nuclear localization of ASF/SF2. While previous studies revealed that SRPK1 phosphorylates RS1, regiospecific and temporal-specific control within the largely redundant RS domain is not well understood. To address this issue, we performed engineered footprinting and single-turnover experiments to determine where and how SRPK1 initiates phosphorylation within the RS domain. The data show that local sequence elements in the RS domain control the strong kinetic preference for RS1 phosphorylation. SRPK1 initiates phosphorylation in a small region of serines (initiation box) in the middle of the RS domain at the C-terminal end of RS1 and then proceeds in an N-terminal direction. This initiation process requires both a viable docking groove in the large lobe of SRPK1 and one RRM (RRM2) on the N-terminal flank of the RS domain. Thus, while local RS/SR content steers regional preferences in the RS domain, distal contacts with SRPK1 guide initiation and directional phosphorylation within these regions.

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Allosteric interactions direct binding and phosphorylation of ASF/SF2 by SRPK1.

Project description:ASF/SF2, a member of the serine-arginine (SR) protein family, has two RRM domains (RRM1 and RRM2) and a C-terminal domain rich in RS dipeptides. SR protein kinase 1 (SRPK1) phosphorylates approximately 12 of these serines using a semiprocessive mechanism. The X-ray structure of the ASF/SF2-SRPK1 complex revealed several features of the complex that raised intriguing questions about how the substrate is phosphorylated by the kinase. The part of the RS domain destined to be phosphorylated at later stages of the reaction docks to a kinase groove distal to the active site while the neighboring RRM2 binds near the active site [Ngo, J. C., et al. (2008) Mol. Cell 29, 563-576]. In this study, we investigate the interplay between the RS domain and RRM2 for stable association and phosphorylation of ASF/SF2. Despite several contacts in the enzyme-substrate complex, free RRM2 does not bind efficiently to SRPK1 unless the docking groove is occupied by the RS domain. This domain cross-talk enhances the processive phosphorylation of the RS domain. The RRM-SRPK1 contact residues control the folding of a critical beta-strand in RRM2. Unfolding of this structural element may force the N-terminal serines of the RS domain into the active site for sequential phosphorylation. Thus, ASF/SF2 represents a new class of substrates that use unique primary sequence to induce allosteric binding, processive phosphorylation, and product release.

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Splicing kinase SRPK1 conforms to the landscape of its SR protein substrate.

Project description:The splicing function of SR proteins is regulated by multisite phosphorylation of their C-terminal RS (arginine-serine rich) domains. SRPK1 has been shown to phosphorylate the prototype SR protein SRSF1 using a directional mechanism in which 11 serines flanked by arginines are sequentially fed from a docking groove in the large lobe of the kinase domain to the active site. Although this process is expected to operate on lengthy arginine-serine repeats (?8), many SR proteins contain smaller repeats of only 1-4 dipeptides, raising the question of how alternate RS domain configurations are phosphorylated. To address this, we studied a splice variant of Tra2? that contains a C-terminal RS domain with short arginine-serine repeats [Tra2?(?N)]. We showed that SRPK1 selectively phosphorylates several serines near the C-terminus of the RS domain. SRPK1 uses a distributive mechanism for Tra2?(?N) where the rate-limiting step is the dissociation of the protein substrate rather than nucleotide exchange as in the case of SRSF1. Although a functioning docking groove is required for efficient SRSF1 phosphorylation, this conserved structural element is dispensable for Tra2?(?N) phosphorylation. These large shifts in mechanism are likely to account for the slower net turnover rate of Tra2?(?N) compared to SRSF1 and may signal fundamental differences in phosphorylation among SR proteins with distinctive arginine-serine profiles. Overall, these data indicate that SRPK1 conforms to changes in RS domain architecture using a flexible kinetic mechanism and selective usage of a conserved docking groove.

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Project description:The glycolytic enzyme, pyruvate kinase Pyk1 maintains telomere heterochromatin by phosphorylating histone H3T11 (H3pT11), which promotes SIR (silent information regulator) complex binding at telomeres and prevents autophagy-mediated Sir2 degradation. However, the exact action mechanism of H3pT11 is poorly understood. Here, we identify Dot1-catalyzed H3K79 tri-methylation (H3K79me3) as the downstream effector of H3pT11 and uncover how this histone crosstalk regulates autophagy and telomere silencing. Mechanistically, Pyk1-catalyzed H3pT11 directly reduces the binding of Dot1 to chromatin and inhibits Dot1-catalyzed H3K79me3, which leads to transcriptional repression of autophagy genes and reduced autophagy. Despite the antagonism between H3pT11 and H3K79me3, they synergically promote the binding of SIR complex at telomeres to maintain telomere silencing. Furthermore, we identify Reb1 as a telomere-associated factor that recruits Pyk1-containing SESAME (Serine-responsive SAM-containing Metabolic Enzyme) complex to telomere regions to phosphorylate H3T11 and prevent the invasion of H3K79me3 from euchromatin into heterochromatin to maintain telomere silencing. Together, these results uncover a novel histone crosstalk and provide insights into dynamic regulation of silent heterochromatin and autophagy in response to cell metabolism.

2022-11-29 | GSE210908 | GEO

Regulating SR protein phosphorylation through regions outside the kinase domain of SRPK1.

Project description:SR proteins (splicing factors containing arginine-serine repeats) are essential splicing factors whose phosphorylation by the SR-specific protein kinase (SRPK) family regulates nuclear localization and mRNA processing activity. In addition to an N-terminal extension with unknown function, SRPKs contain a large, nonhomologous spacer insert domain (SID) that bifurcates the kinase domain and anchors the kinase in the cytoplasm through interactions with chaperones. While structures for the kinase domain are now available, constructs that include regions outside this domain have been resistant to crystallographic elucidation. To investigate the conformation of the full-length kinase and the functional role of noncatalytic regions, we performed hydrogen-deuterium exchange and steady-state kinetic experiments on SRPK1. Unlike the kinase core, the large SID lacks stable, hydrogen-bonded structure and may provide an intrinsically disordered region for chaperone interactions. Conversely, the N-terminus, which positively regulates SR protein binding, adopts a stable structure when the insert domain is present and stabilizes a docking groove in the large lobe of the kinase domain. The N-terminus and SID equally enhance SR protein turnover by altering the stability of several catalytic loop segments. These studies reveal that SRPK1 uses an N-terminal extension and a large, intrinsically disordered region juxtaposed to a stable structure to facilitate high-affinity SR protein interactions and phosphorylation rates.

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