Analysis of Cbf5-dependent mRNA pseudouridylation and its function in the mRNA life cycle

Abstract

Chemical nucleotide modifications of (ribonucleic acids) RNAs take center stage of the current research. The isomerization of a uridine to a pseudouridine, known as pseudouridylation, is the most abundant RNA modification. One pseudouridine synthase that catalyzes pseudouridylation of ribosomal RNAs (rRNAs) is Cbf5 in Saccharomyces cerevisiae. Cbf5 harbors the catalytic activity of the H/ACA small nucleolar ribonucleoprotein particle (H/ACA snoRNP), which also contains the proteins Gar1, Nhp2 and Nop10 and one guide H/ACA small nucleolar RNA (H/ACA snoRNA). In 2014/15, transcriptome-wide mapping of pseudouridylation sites showed that Cbf5 also pseudouridylates residues of messenger RNAs (mRNAs). So far, little is known about the prevalence, mechanism and function of Cbf5-dependent mRNA pseudouridylation and the role of Cbf5 in the mRNA life cycle. Previous results revealed an RNA-independent interaction between Cbf5 and TREX. TREX (TRanscription and EXport) is a conserved nuclear protein complex, which has a central role in coordination of mRNA synthesis and nuclear mRNA export. In this study it was shown that Cbf5 also functions in transcription and nuclear mRNA export. Fluorescence in situ hybridizations (FISHs) demonstrated an mRNA export defect when Cbf5 is depleted. In addition, Cbf5 influences transcription elongation of the RNA polymerase II (RNAPII) and it also controls transcription of genes involved in metabolism and energy derivation shown by RNAPII chromatin immunoprecipitation sequencing (ChIPseq). Cbf5-dependent pseudouridylation in the 5’ untranslated region (UTR) of the transcription activator GCN4 regulates its protein amount that might influence transcription of genes involved in amino acid metabolism. Moreover, a point mutation at the Cbf5-dependent pseudouridylation site of ADE13 regulates mRNA synthesis of this gene. Additionally, it was shown that also Gar1 and Nhp2 of the H/ACA snoRNP interact with TREX in an RNA-independent manner. Chromatin immunoprecipitations (ChIPs) and RNA immunoprecipitations (RIPs) demonstrate that Cbf5 is present at all genes and mRNAs, independent of whether the mRNA has a pseudouridylation site. Furthermore, it was shown that Cbf5 occupancy at the genes is transcription- and RNA-dependent. Primer extension after N-cyclohexyl-N9-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulphonate (CMC) labeling of nascent and poly(A) mRNA suggests that Cbf5-dependent pseudouridylation occurs post-transcriptionally. To summarize, it was shown for the first time that Cbf5 has a role in mRNA synthesis and nuclear mRNA export just as the TREX complex. Whereas the role of Cbf5 in transcription is pseudouridylation-dependent, FISHs demonstrated that the nuclear mRNA export function of Cbf5 is pseudouridylation-independent.