Functional interaction of human Ssu72 with RNA polymerase II complexes

Abstract

Phosphorylation of the C-terminal domain (CTD) of the large subunit of human RNA polymerase II (Pol II) is regulated during the transcription cycle by the combined action of specific kinases and phosphatases. Pol II enters into the preinitiation complex (PIC) unphosphorylated, but is quickly phosphorylated by Cdk7 during initiation. How phosphatases alter the pattern and extent of CTD phosphorylation at this early stage of transcription is not clear. We previously demonstrated the functional association of an early-acting, magnesium-independent phosphatase with early elongation complexes. Here we show that Ssu72 is responsible for that activity. We found that the phosphatase enters the transcription cycle during the formation of PICs and that Ssu72 is physically associated with very early elongation complexes. The association of Ssu72 with elongation complexes was stable to extensive washing with up to 200 mM KCl. Interestingly, Ssu72 ceased to function on complexes that contained RNA longer than 28 nt. However, when PICs were washed before initiation, the strict cutoff at 28 nt was lost. This suggests that factor(s) are important for the specific regulation of Ssu72 function during the transition between initiation and pausing. Overall, our results demonstrate when Ssu72 can act on early transcription complexes and suggest that Ssu72 may also function in the PIC prior to initiation.

Bioinformatic Identification of Chinese Hamster Ovary (CHO) Cold‐Shock Genes and Biological Evidence of their Cold‐Inducible Promoters

“…Some of the top ten identified genes could be related to stress response, mainly Txnip, Angplt4, and Gstm7, but also S100a6 through inhibition of the interaction between heat shock proteins and Sgt1 or Hop . On the other hand, the highest ranked gene from the list, SSu72, plays a role in RNA processing and termination and could be related to the increase in transcription . To confirm and better understand the link between these genes and cold response in CHO cells would require further studies.…”

“…[35][36][37]39] On the other hand, the highest ranked gene from the list, SSu72, plays a role in RNA processing and termination and could be related to the increase in transcription. [33] To confirm and better understand the link between these genes and cold response in CHO cells would require further studies.…”

Identification of Novel MeCP2 Cancer-Associated Target Genes and Post-Translational Modifications

“…In MeCP2 ChIP-Seq in MDA-MB-468 cells, we found that MeCP2 binds to multiple novel targets not previously associated with MeCP2 function in the context of breast cancer. Some of these included the following genes: a) SSU72 Homolog, RNA Polymerase II CTD Phosphatase (SSU72), a protein phosphatase that catalyzes the dephosphorylation of the C-terminal domain of RNA polymerase II ( 66 ); b) CAPN2 (Calpain 2), a calcium-sensitive cysteine protease ( 67 ); c) Plexin B2 (PLXNB2), a class B transmembrane receptor that participates in axon guidance and cell migration in response to semaphorins ( 68 ); d) Zinc Finger SWIM-Type Containing 4 (ZSWIM4); e) RUNX Family Transcription Factor 3 (RUNX3) a transcription factor that functions as a tumor suppressor and is frequently deleted or transcriptionally silenced in cancer ( 69 , 70 ), and f) Solute Carrier Family 45 Member 4 (SLC45A4) ( Figure 1B ). Additionally, in MCF7 some of the notable genes included a) Ubiquitin Specific Peptidase 34 (USP34), a ubiquitin hydrolase that removes conjugated ubiquitin from AXIN1 and AXIN2, acting as a regulator of Wnt signaling pathway ( 71 ); b) Maltase–Glucoamylase (MGAM), an enzyme that plays a role in the digestion of starch ( 72 ); c) GDP-Mannose 4,6-Dehydratase (GMDS), an enzyme that participates in the synthesis of GDP-fucose from GDP-mannose ( 73 ); d) Solute Carrier Family 45 Member 4 (SLC45A4); e) CCDC26 Long Non-Coding RNA (CCDC26), a lncRNA class associated with Malignant Glioma and Astrocytoma ( 74 , 75 ); and f) Sidekick Cell Adhesion Molecule 1 (SDK1) ( Figure 1C ).…”

Ssu72: a versatile protein with functions in transcription and beyond

“…An analysis of the genetic and physical interactions of Ssu72 with the promoter-bound factors implicated the protein in the assembly of the preinitiation complex (PIC) ( Ganem et al, 2003 ; Spector et al, 2019 ). Prior to initiation of transcription, the preinitiation complex (PIC) recruits RNAPII with a hypo-phosphorylated CTD.…”