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DYRK1A's specificity in divergent CTD heptads (A) Diagram of the CTD sequence of human RPB1. The shaded region consists of the consensus sequence. The rest of the sequence diverges slightly from consensus, with residues different from serine in the seventh position highlighted. (B) Structural model of DYRK1A in complex with a CTD peptide where the seventh position is occupied by arginine. DYRK1A is shown in the ribbon diagram as light pink, and the CTD peptide as sticks is shown as light blue. Hydrophilic interactions are denoted with dashed lines. (C-F) Chromatographic traces for the LC-MS/MS analysis of DYRK1A's specificity toward CTD peptides containing three heptads. Gold-colored LC traces correspond to the unphosphorylated peptide, whereas the purple traces indicate the monophosphorylated species with peak numbers matching the sites of phosphorylation indicated on the sequences above the LC traces. (C) the middle heptad containing arginine in the seventh position. (D) the middle heptad containing lysine in the seventh position. (E) each heptad containing glutamate in the seventh position. (F) each heptad containing glutamine in the seventh position.
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During eukaryotic transcription, RNA polymerase II undergoes dynamic post-translational modifications on the C-terminal domain (CTD) of the largest subunit, generating an information-rich PTM landscape that transcriptional regulators bind. The phosphorylation of Ser5 and Ser2 of CTD heptad occurs spatiotemporally with the transcriptional stages, re...
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... active site interactions of DYRK1A (light blue) with the modeled CTD peptide (salmon) are highlighted with key residues shown in stick and hydrogen bonds in dash lines. iScience Article we used P-TEFb to treat the CTD heptad sequence, the only phosphorylation species was at Ser5 ( Figures 1C and S2). Previous studies using tandem mass spectrometry confirmed that P-TEFb phosphorylates Ser2 when the CTD is primed by Tyr1 phosphorylation; otherwise, the phosphorylation occurs at Ser5. 19 Other CDKs also preferentially phosphorylate Ser5 over Ser2 with conserved recognition structural motifs. ...
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... reduction of phosphorylation in both Ser2 and Ser5 upon DYRK1A knockdown was previously reported. 20 To investigate the possibility of alternative sites being phosphorylated with different sequence contexts, we systematically characterized DYRK1A's specificity using biochemical product profiling with high-resolution mass spectrometric characterization (Figure 2). With the 7 th residue in the previous heptad ...
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... 26, 107581, September 15, 2023identified as key to DYRK1A ( Figure 1E), we systematically replaced the preceding 7 th residue and characterized the product phosphorylation sites (Figure 2). Human RNA Pol II is enriched with sequences divergent from the consensus at the 7 th position of the heptad, where the most frequent replacements are positively charged residues like Lys and Arg ( Figure 2A). ...
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... 26, 107581, September 15, 2023identified as key to DYRK1A ( Figure 1E), we systematically replaced the preceding 7 th residue and characterized the product phosphorylation sites (Figure 2). Human RNA Pol II is enriched with sequences divergent from the consensus at the 7 th position of the heptad, where the most frequent replacements are positively charged residues like Lys and Arg ( Figure 2A). Our binding model of DYRK1A for Ser2 phosphorylation places these positively charged residues along with Arg323 and Arg327 to stabilize the activating residue phosphoryl-Tyr321 ( Figure 2B). ...
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... RNA Pol II is enriched with sequences divergent from the consensus at the 7 th position of the heptad, where the most frequent replacements are positively charged residues like Lys and Arg ( Figure 2A). Our binding model of DYRK1A for Ser2 phosphorylation places these positively charged residues along with Arg323 and Arg327 to stabilize the activating residue phosphoryl-Tyr321 ( Figure 2B). To test this structural prediction, we used a substrate CTD with the 7 th residue as Arg or Lys in the proceeding heptad; the Ser2 in the subsequent heptad gets phosphorylated preferably and effectively ( Figures 2C, 2D, S4, and S5). ...
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... binding model of DYRK1A for Ser2 phosphorylation places these positively charged residues along with Arg323 and Arg327 to stabilize the activating residue phosphoryl-Tyr321 ( Figure 2B). To test this structural prediction, we used a substrate CTD with the 7 th residue as Arg or Lys in the proceeding heptad; the Ser2 in the subsequent heptad gets phosphorylated preferably and effectively ( Figures 2C, 2D, S4, and S5). Favorable interactions of Lys7/Arg7 with DYRK1A place the neighboring Ser2 at a highly favorable position for kinase phosphorylation ( Figures 2C and 2D). ...
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... test this structural prediction, we used a substrate CTD with the 7 th residue as Arg or Lys in the proceeding heptad; the Ser2 in the subsequent heptad gets phosphorylated preferably and effectively ( Figures 2C, 2D, S4, and S5). Favorable interactions of Lys7/Arg7 with DYRK1A place the neighboring Ser2 at a highly favorable position for kinase phosphorylation ( Figures 2C and 2D). In contrast, if a negatively charged residue like glutamate or phosphorylated Ser7 occupies the 7 th residue, it is positioned too close to the phosphoryl-Tyr of DYRK1A. ...
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... unfavorable repelling interaction switches the mode of substrate recognition. Indeed, we previously noticed Ser5 phosphorylation as the major product when we used heptad repeats containing E at the 7 th position ( Figure 2E and S6). 21 Furthermore, due to space limitations, bulky residues like glutamine in this 7 th position also hamper the hydrogen bond network with DYRK1A. ...
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... Furthermore, due to space limitations, bulky residues like glutamine in this 7 th position also hamper the hydrogen bond network with DYRK1A. The exclusion of Ser2 binding mode leads to Ser5 on the heptad being phosphorylated with no phosphoryl-Ser2 detected ( Figures 2F and S7). Therefore, our mass spectrometric and structural analyses reveal that DYRK1A strongly prefers the phosphorylation of CTD at the Ser2 position when the 7 th residue from the preceding heptad is occupied by a small polar or positively charged residue. ...
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... investigate the role of CHERP in transcription, we performed RNA-seq analysis to measure the polyadenylated mRNA in cells where the CHERP protein is knocked down. As evaluated in the Western blot, we used a commercially available shRNA to reduce the CHERP expression to 18% ( Figure S12D). We then conducted deep sequencing of the mRNA with CHERP knocked down compared to the wild type. ...
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... then conducted deep sequencing of the mRNA with CHERP knocked down compared to the wild type. Correlation analysis showed that both biological replicates clustered depending on the condition and strongly correlated with each other (r > 0.99) (Figures S12A-S12C). We found that the overall transcriptome was not significantly affected by the loss of CHERP. ...
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... identified 2,135 AS events in 1,560 unique genes upon CHERP inhibition vs. Control (FDR <0.05, ILD, inclusion level difference, R10%) ( Figure S12E). Based on GO analysis, alternatively, spliced transcripts were enriched for proteins related to cilium organization and cell polarity, cell cycle G2/M transition, response to endoplasmic reticulum stress, and DNA damage checkpoint ( Figure 6C; and Table S3). ...
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Citations
The RNA polymerase II carboxy-terminal domain (CTD) consists of conserved heptapeptide repeats that can be phosphorylated to influence distinct stages of the transcription cycle, including RNA processing. Although CTD-associated proteins have been identified, phospho-dependent CTD interactions have remained elusive. Proximity-dependent biotinylation (PDB) has recently emerged as an alternative approach to identify protein-protein associations in the native cellular environment. In this study, we present a PDB-based map of the fission yeast RNAPII CTD interactome in living cells and identify phospho-dependent CTD interactions by using a mutant in which Ser2 was replaced by alanine in every repeat of the fission yeast CTD. This approach revealed that CTD Ser2 phosphorylation is critical for the association between RNAPII and the histone methyltransferase Set2 during transcription elongation, but is not required for 3′ end processing and transcription termination. Accordingly, loss of CTD Ser2 phosphorylation causes a global increase in antisense transcription, correlating with elevated histone acetylation in gene bodies. Our findings reveal that the fundamental role of CTD Ser2 phosphorylation is to establish a chromatin-based repressive state that prevents cryptic intragenic transcription initiation.
Decades of scientific research have been devoted to unraveling the intricacies of eukaryotic transcription since the groundbreaking discovery of eukaryotic RNA polymerases in the late 1960s. RNA polymerase II, the polymerase responsible for mRNA synthesis, has always attracted the most attention. Despite its structural resemblance to its bacterial counterpart, eukaryotic RNA polymerase II faces a unique challenge in progressing transcription due to the presence of nucleosomes that package DNA in the nuclei. In this review, we delve into the impact of RNA polymerase II and histone signaling on the progression of eukaryotic transcription. We explore the pivotal points of interactions that bridge the RNA polymerase II and histone signaling systems. Finally, we present an analysis of recent cryo-electron microscopy structures, which captured RNA polymerase II-nucleosome complexes at different stages of the transcription cycle. The combination of the signaling crosstalk and the direct visualization of RNA polymerase II-nucleosome complexes provides a deeper understanding of the communication between these two major players in eukaryotic transcription.
