A novel assay identifies transcript elongation roles for the Nup84 complex and RNA processing factors

• Published in: The EMBO journal Vol. 30; no. 10; pp. 1953 - 1964
• Main Authors: Tous, Cristina, Rondón, Ana G, García-Rubio, María, González-Aguilera, Cristina, Luna, Rosa, Aguilera, Andrés
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 18.05.2011; Nature Publishing Group UK; Blackwell Publishing Ltd; Nature Publishing Group
• Subjects: Bioassays; Chromatin; Chromatin Immunoprecipitation; Deoxyribonucleic acid; DNA; DNA Transposable Elements; EMBO09; G-less run-on; Gene Knockout Techniques; Molecular biology; Molecular Biology - methods; mRNA factors; Mutagenesis, Insertional; Nuclear Pore Complex Proteins - genetics; Nuclear Pore Complex Proteins - metabolism; Nup84; Ribonucleic acid; RNA; RNA, Fungal - metabolism; RNA, Messenger - metabolism; RNAPII; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - physiology; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Substrates; transcription elongation; Transcription, Genetic; G‐less run‐on; transcription elongation; mRNA factors; RNAPII; Nup84
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1038/emboj.2011.109

To clarify the role of a number of mRNA processing factors in transcription elongation, we developed an in vivo assay for direct analysis of elongation on chromatin. The assay relies on two substrates containing two G‐less cassettes separated by either a long and GC‐rich or a short and GC‐poor DNA sequence (G‐less‐based run‐on (GLRO) assay). We demonstrate that PAF, THSC/TREX‐2, SAGA, the exosome component Rrp6 and two subunits of cleavage factor IA (Rna14 and Rna15) are required for efficient transcription elongation, in contrast to some results obtained using other assays. Next, we undertook a mutant screen and found out that the Nup84 nucleoporin complex is also required for transcription elongation, as confirmed by the GLRO assay and RNA polymerase II chromatin immunoprecipitations. Therefore, in addition to showing that the GLRO assay is a sensitive and reliable method for the analysis of elongation in vivo , this study provides evidence for a new role of the Nup84 complex and a number of mRNA processing factors in transcription elongation that supports a connection of pre‐mRNA processing and nuclear export with transcription elongation. This new in vivo G‐less‐based run‐on assay (GLRO) identifies roles for several RNA processing factors and Nup84 in directly regulating transcriptional elongation.