| RNA Polymerase Ⅱ(RNAPⅡ)is one of the most important complexes in eukaryotes.It consists of 12 subunits and is responsible for synthesizing all mRNA and some non-coding RNA.So far,the structure and function of RNAPII are well understood,but the molecular assembly mechanism of its 12 subunits is not clear.Prokaryotic RNA polymerase can be assembled spontaneously in vitro,while eukaryotic RNA polymerase cannot,indicating that the assembly process of eukaryotic RNA polymerase requires the participation of assembly factors.RNAPⅡ assembly factors have been identified in Saccharomyces cerevisiae,including Rba50,Npa3,Gpn2,Gpn3,Rtr1,Bud27,which are all highly conserved proteins.A new phenomenon has been found that prolonged inactivation of the RNAPⅡassembly factor causes the RNAPⅡI subunit to reside in the cytoplasm and form condensates.The Condensates were named RNA Polymerase Ⅱ Subunit Cytoplasmic Condensates(RSCCs).Many different types of condensates have been found in eukaryotic cells and it is generally believed that phase separation is one of the important mechanisms of condensate formation.It is speculated that RSCCs may be a novel phase separation structure.To determine whether the mechanism of RSCCs formation is similar to that of classical cytoplasmic condensates found in the cytoplasm.We observed the localization of RSCCs and SGs(Stress granules),P body(Processing body),and PSG(Proteasome storage granule)granule in cells,and the results showed that RSCCs and SGs granule were colocated.Some particles of RSCCs and PSG are co-located,while RSCCs and P Body are not co-located.It is concluded that there may be common components and similar formation mechanisms between RSCCs and SGs,and PSG may be involved in ubiquitination and degradation of RSCCs,while P body may be unrelated to the formation of RSCCs.In order to further reveal the mechanism of RSCCs formation,it is necessary to find the key regulatory factors of RSCCs formation.In this study,two methods were designed to identify the key regulatory factors of RSCCs formation.One was to use the temperature-sensitive mutant gpn3-9 to inactivate the assembly factor Gpn3 at a limited temperature.Precipitation of the RNAPII subunit interacting proteins was evaluated using Co-IP(Co-inmunoprecipitation)combined with MS(Mass spectrography).The results showed that Hsp82 may be a key regulatory protein in the formation of RSCCs,and fluorescence observation showed that Hsp82 colocalized with RSCCs.Second,AID targeted degradation technology was used to target degradation assembly factor Gpn3,and Co-IP combined with MS was used to identify novel proteins interacting with RNAPⅡsubunit.After targeted degradation of Gpn3,new proteins interacting with Rpb1,Rpb2 and Rpb3 were screened,including candidate regulatory proteins Ctr9,Not5,Yta12,Cdc19 and Rrm3.In order to directly obtain RSCCs and understand their components,this project has established a RSCCs particle separation and purification system,which can isolate and purify RSCCs efficiently.Then,the isolated and purified RSCCs were identified by Co-IP combined with MS,and it was determined that most of RNAPⅡ subunits and some other component proteins were contained in the condensate.Some of the components were further verified,and the co-localization of the chaperone protein Ssal and RSCCs was confirmed.The RSCCs were treated by DNase I,RNase A and Proteinase K,and it was found that RSCCs were protein condensates without DNA and RNA components.At the same time,the phase separation characteristics of RSCC are analyzed.RSCCs,P body and SGs were treated with 1,6 hexadiol at the same time.It was found that P body was completely degraded,while RSCCs and SGs were weakly sensitive.This indicates that RSCCs may have a similar phase separation characteristic to SGs,which is different from P body.The rapid recovery of RSCCs after partial fluorescence bleaching proves that RSCCs are highly dynamic phase separation structures.The characteristic sequences of phase separation in 12 subunits of RNAPⅡ were predicted and analyzed..It was found that Rpbl,Rpb2,Rpb3,Rpb6,Rpb9,Rpb10,Rpb11 and Rpb12 had obvious characteristic sequences required for the formation of phase separation,which may play an important role in the formation of condensates.These results advance the study of the assembly mechanism of eukaryotic RNA polymerase Ⅱ and provide a new understanding of transcriptional regulation in eukaryotes. |
