Rtr1 is required for Rpb1-Rpb2 assembly of RNAPII and prevents their cytoplasmic clump formation.

RNA polymerase II (RNAPII) is an essential machinery for catalyzing mRNA synthesis and controlling cell fate in eukaryotes. Although the structure and function of RNAPII have been relatively defined, the molecular mechanism of its assembly process is not clear. The identification and functional analysis of assembly factors will provide new understanding to transcription regulation. In this study, we identify that RTR1, a known transcription regulator, is a new multicopy genetic suppressor of mutants of assembly factors Gpn3, Gpn2, and Rba50. We demonstrate that Rtr1 is directly required to assemble the two largest subunits of RNAPII by coordinating with Gpn3 and Npa3. Deletion of RTR1 leads to cytoplasmic clumping of RNAPII subunit and multiple copies of RTR1 can inhibit the formation of cytoplasmic clump of RNAPII subunit in gpn3-9 mutant, indicating a new layer function of Rtr1 in checking proper assembly of RNAPII. In addition, we find that disrupted activity of Rtr1 phosphatase does not trigger the formation of cytoplasmic clump of RNAPII subunit in a catalytically inactive mutant of RTR1. Based on these results, we conclude that Rtr1 cooperates with Gpn3 and Npa3 to assemble RNAPII core.

Npa3-Gpn3 cooperate to assemble RNA polymerase II and prevent clump of its subunits in the cytoplasm.

RNA polymerase II (RNAPII) is an essential machinery in eukaryotes that catalyzes mRNA synthesis and controls cell fate. Although the structure and function of RNAPII are relatively well defined, the molecular mechanism of its assembly process is poorly understood. Three members of GPN-loop GTPase family Npa3/Gpn1, Gpn2, and Gpn3 participate in the biogenesis of RNAPII with non-redundant roles. In this study, we demonstrate that Gpn3 and Npa3 directly participate in the assembly of the two largest subunits during biogenesis of RNAPII. When Gpn3 is defective, assembly of RNAPII is disrupted, leading to cytoplasmic foci of RNAPII subunits. Long-term assembly factor defects will lead to the accumulation of different kind of newly synthesized RNAPII subunits in the cytoplasm to form foci, and this can be prevented by recovery of the defective assembly factor. Cytoplasmic foci of RNAPII subunits in mutants of these assembly factors reveals a new cellular rescue response named the 'RNAPII assembly stress response'.

Rba50 and Gpn2 recruit the second largest subunits for the assembly of RNA polymerase II and III.

Although remarkable progress has been made toward understanding the structures of eukaryotic RNA polymerases, the pathways and factors that facilitate their assembly remain unresolved. Essential proteins Rba50 and Gpn2 are required for Rpb3 subcomplex assembly, but whether they participate in subsequent assembly steps is unknown. Herein, we performed comprehensive genetic screens to explore Rba50 function. We identified two unique extragenic rba50-3-suppressing mutations that map to genes encoding the Rba50-interacting protein Gpn2, and Rpb2, the second largest subunit of RNAPII. Both gpn2-R347S and rpb2-V1171G variants bypass Rpb1 cytoplasmic arrest and temperature-sensitive growth defects of the rba50-3 mutant. GPN2 and RPB2 were also identified as novel multicopy suppressors of the rba50-3 mutant. Rapid depletion of Rba50 affected Rpb3-Rpb2 association during RNAPII assembly. Importantly, we demonstrated that Gpn2 facilitates the association of Rba50 and Rpb2. Our results imply that Rba50-Gpn2 interaction is essential for Rpb2 recruitment during RNAPII assembly following Rpb3 subcomplex assembly. Furthermore, the Rba50-Gpn2 complex appears to play a similar role in the assembly of RNAPIII. We therefore propose a model in which Rba50 interacts with Gpn2 and thereby promotes loading of the second largest subunit of RNAP II and III onto the previously assembled subcomplex.

Npa3 interacts with Gpn3 and assembly factor Rba50 for RNA polymerase II biogenesis.

RNA polymerase II is one of the most vital macromolecular complexes in eukaryotes and the assembly of such complete enzyme requires many factors. Three members of GPN-loop GTPase family Npa3/Gpn1, Gpn2, and Gpn3 participate in the biogenesis of RNA polymerase II with nonredundant roles. We show here that rapid degradation of each GPN protein in yeast leads to cytoplasmic accumulation of Rpb1 and defects in the assembly of RNA polymerase II, suggesting conserved functions of GPN paralogs for RNA polymerase II biogenesis as in humans. Taking advantage of a multicopy genetic screening, we identified GPN3 and assembly factor RBA50 among others as strong suppressors of npa3ts mutants. We further demonstrated that Npa3 interacts with Gpn3 and Rba50, similarly human Gpn1 physically interacts with Gpn3 and RPAP1 (human analog of Rba50). Moreover, a mutual dependency of protein levels of Npa3 and Gpn3 was also clearly presented in yeast using an auxin-inducible degron (AID) system. Interestingly, Rpb2, the second largest subunit of RNA polymerase II was determined to be the subunit that interacts with both Gpn1 and Rba50, indicating a close association of Npa3 and Rba50 in Rpb2 subcomplex assembly. Based on these results, we conclude that Npa3 interacts with Gpn3 and Rba50, for RNA polymerase II biogenesis. We therefore propose that multiple factors may coordinate through conserved regulatory mechanisms in the assembly of RNA polymerase complex.

Dual Repressive Function by Cip1, a Budding Yeast Analog of p21, in Cell-Cycle START Regulation.

Cip1, a newly identified yeast analog of p21, is a Cln3-CDK inhibitor that negatively regulates cell-cycle START. However, its function remains poorly understood. In this study, we found that deletion of CLN3 did not result in bypass of G1-phase arrest caused by Cip1 overexpression. Cip1 depletion in cln3-null mutants significantly advanced the timing of Cln2 expression, supporting the idea that Cip1 represses START in a Cln3-independent manner. We set to search for novel Cip1 interacting proteins and found that Ccr4, a known START regulator, and its associated factor Caf120, interact with Cip1. Ccr4-Caf120 acts redundantly with Cdk1-Cln3 to inhibit Whi5-mediated regulation of START. This interaction was conserved between human Ccr4 and p21. In addition, deletion of WHI5 robustly suppressed G1-phase arrest caused by Cip1 overexpression. We conclude that Cip1 negatively regulates START by acting as a dual repressor of Ccr4 in parallel with Cln3.

An optimized yeast display strategy for efficient scFv antibody selection using ribosomal skipping system and thermo resistant yeast.

OBJECTIVES: Establish a method to restrict unexpected fragments including stop codons in scFv library and generate a thermo resistant strain for screening of thermal stable scFv sequences. RESULTS: Here, we have constructed a T2A-Leu2 system for selection of yeast surface display libraries that blocks amplification of "stop codon" plasmids within the library, thereby increasing the quality of the library and efficiency of the selection screen. Also, we generated a temperature-resistant yeast strain, TR1, and validated its combined use with T2A-Leu2 for efficient screening. Thus, we developed a general approach for a fast and efficient screening of scFv libraries using a ribosomal skipping system and thermo-resistant yeast. CONCLUSIONS: The method highlights the utility of the T2A-Leu2-based ribosomal skipping strategy for increasing the quality of the input library for selection, along with an optimized selection protocol based on thermo-resistant yeast cells.