RNA Polymerase III transcription initiation

        

Eukaryotic cells use different forms of RNA polymerase (Pol) for the transcription of their genomes. These RNA polymerases have similar architectures, based on a set of ten evolutionarily conserved subunits that form the core enzyme. Three distinct promoter types direct transcription by human Pol III (hPol III), producing short, untranslated transcripts including tRNA and 5S rRNA5. The initial recognition event is sufficient to recruit TFIIIB and then Pol III to the promoter, altogether forming a pre-initiation complex competent for ATP-independent melting of promoter DNA.

RNA polymerase III is the largest of the eukaryotic RNA polymerases. Two-hybrid screenings have established a detailed description of the protein-protein interaction network of Pol III subunits and transcription factors. Five Pol III–specific subunits do not have structurally equivalent or evolutionarily related subunits in the other RNA polymerases. Of these subunits, RPC62, RPC39 and RPC32 associate to form a stable subcomplex. This complex is crucial for specific transcription initiation at Pol III promoters, although Pol III lacking this ternary complex remains competent in transcription, elongation and termination.

It has been demonstrated that deletion of the C-terminal region of RPC32 impairs selective transcription initiation from Pol III promoters but not from a nonspecific template, and does not affect the catalytic properties of the enzyme. Two isoforms of RPC32 have been described whose expression is regulated (RPC32 alpha)  or constitutively expressed (RPC32 beta)  that yield the two Pol III isoforms. RPC39 also play a role in the initiation step of transcription through their direct interaction with Brf1 and by contributing to promoter opening. 

We solved the structure of RPC62  and RPC62 in complex with RPC32 beta and demonstrated that core-subnit of Pol III share structural similarity with Pol II transcription factors, bringing an unforeseen link between the three nuclear RNA polymerases.

                             

               

 

Ogunjimi et al. Inborn errors in RNA polymerase III underlie severe varicella zoster virus infections (2017) J. Clin. Invest.,127(9), 3543-3556. doi:10.1172/JCI92280.

Thiffault et al. Recessive mutations in POLR1C cause a leukodystrophy by impairing biogenesis of RNA polymerase III (2015) Nat. Comm, 6, 7623.

F. Boissier, H. Dumay-Odelot, M. Teichmann, S. Fribourg. Structural analysis of RPC32b - RPC62 complex (2015) J. Struct. Biol, 192, 313-319.

N. Wolf, A. Vanderver, R. van Spaendonk, R. Schiffmann, B. Brais, M. Bugiani, E. Sistermans, C. Catsman-Berrevoets, J. Kros, P. Soares Pinto, D. Pohl, S. Tirupathi, P. Stromme, T. de Grauw, S. Fribourg, M. Demos, A. Pizzino, S. Naidu, K. Guerrero, M. S. van der Knaap, and G. Bernard. Clinical spectrum of 4H leukodystrophy caused by POLR3A and POLR3B mutations (2014) Neurology, 18, 1898-1905

Teichmann M., Dumay-Odelot H., Fribourg S. (2012). Structural and functional aspects of winged-helix domains at the core of transcription initiation complexes. Transcription, 3, 2-7.

Lefevre S, Dumay-Odelot H, El Ayoubi L, Budd A, Legrand P, Pinaud N, Teichmann M, Fribourg S (2011) Structure-function analysis of hRPC62 provides insights into RNA polymerase III transcription initiation. Nat. Struct. Mol. Biol., 18(3), 352-358.

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Crystal Structure of human RPC62 subunit

Lefèvre et al. Nat. Struct. Mol Biol. (2011)

Crystal Structure of human RPC62 and RPC32 beta heterodimer

Boisser et al. J. Stuct. Biol. (2015)

Diamond-Blackfan anemia

 

Diamond–Blackfan anemia (DBA) is a rare congenital disorder characterized by the defective differentiation of pro-erythroblasts, the precursors of red blood cells. Patients suffer from  a severe anemia and display heterogeneous clinical features including malformations, growth failure and predisposition to cancer. Linkage analysis has revealed that a quarter of all DBA reported cases are connected to the heterozygous mutation of the gene encoding the ribosomal protein RPS19. The RPS19 protein is a component of the 40S ribosomal subunit and belongs to a family of ribosomal proteins restricted to eukaryotes and archea. It is essential for yeast viability and for early stages of development in mice. Disruption as well as point mutations of the rps19 gene in yeast and human cells affect maturation of the pre-ribosomal RNA (pre-rRNA) and block the production of 40S ribosomal subunits. Why the mutation of a ribosomal protein primarily affects pro-erythroblast differentiation remains a central question. Recent linkage of ribosomal protein genes such as rps7, rps10, rps24, rps17, rps26,  rps27, rps28, rpl5, rpl11, rpl15, rpl35a, rpl36, rpl26, , rpl27, tsr2 and gata1 to DBA strongly supports the hypothesis that DBA is a ribosomal disorder. Over 60 different mutations affecting the rps19 gene have been reported, including deletions, insertions, frameshifts, pre-mature stop codons and missense mutations. Some mutations, like very early stop codons or modification of the promoter clearly result in RPS19 haploinsufficiency by hampering synthesis of RPS19 from the mutated allele. However, for more subtle mutations like mis-sense mutations, the question arises as to whether they affect the folding of the protein or whether they are milder mutations affecting the function while preserving the overall fold. 

-Aguissa-Touré AH, Da Costa L, Leblanc T, Tchernia G, Fribourg S, Gleizes PE (2009) Med Sci., 25, 69-76.

-Choesmel V, Fribourg S, Aguissa-Touré AH, Pinaud N, Legrand P, Gazda HT & Gleizes PE. (2008) Hum Mol Genet.,17, 1253-1263 .

 

-Gregory L.A., Aguissa-Touré A.H., Legrand P., Pinaud N., Gleizes P.E. & Fribourg S. (2007) Nucl. Acids Res., 35, 5913-5921.

 

-Choesmel V, Bacqueville D, Rouquette J, Noaillac-Depeyre J, Fribourg S, Cretien A, Leblanc T, Tchernia G, Da Costa L,  & Gleizes PE. (2007) Blood,109, 1275-1263 .

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