Yeast Pre-rRNA Processing and Modification Occur Cotranscriptionally

Abstract

SummaryTo better understand yeast ribosome synthesis, we developed techniques for the rapid harvesting and analysis of metabolically labeled cultures. Modeling of the resulting kinetic data allowed predicted lifetimes and processing patterns to be compared with the experimental data. This supported a transcription time for the 35S primary transcripts of ∼170 s at 30°C (∼40 nt s−1), with a high fraction (∼70%) of nascent transcripts cleaved at the early processing sites that generate the 20S precursor to the 18S rRNA. This level of nascent transcript cleavage apparently conflicted with previous reports that modification of yeast pre-rRNA exclusively occurred on released transcripts. A second round of high-resolution kinetic labeling showed that 20S pre-rRNA predominately undergoes methylation as nascent transcripts, whereas the 27S precursor to the 25S/5.8S rRNAs was partially methylated on the nascent transcript. The results demonstrate that quantitative analyses of pre-rRNA processing can yield important biological insights.

Ribosomal proteins L7 and L8 function in concert with six A3 assembly factors to propagate assembly of domains I and II of 25S rRNA in yeast 60S ribosomal subunits

“…Greater amounts of A 3 ends relative to A 2 and B 1S ends observed upon depletion of L7 or L8 might also result from a switch from cotranscriptional to post-transcriptional processing of pre-rRNA (Osheim et al 2004;Koš and Tollervey 2010). The higher levels of 23S pre-rRNA observed in pulse-chase experiments with either depleted strain indicate that the relative rate of production of 27SA 3 increases.…”

“…During and after transcription, precursor-rRNAs (pre-rRNAs) are modified and folded to form structures necessary for removal of transcribed spacer sequences and for binding of the 79 ribosomal proteins (r-proteins) (van Nues et al 1995;Cô té et al 2002;Staley and Woolford 2009;Karbstein 2011). Initial cleavage events within transcribed spacer sequences, some of which occur cotranscriptionally (Osheim et al 2004;Koš and Tollervey 2010), generate 43S and 66S precursors to mature 40S and 60S ribosomal subunits, respectively, which then undergo separate pathways of maturation.…”

“…Although ribosome assembly in vivo is more efficient than in vitro, it is also more complex. First, assembly occurs cotranscriptionally in vivo (Osheim et al 2004;Koš and Tollervey 2010). Proteins bind to rRNA structures that form as the RNA is transcribed, largely in a 59 to 39 direction (Adilakshmi et al 2005;Talkington et al 2005;Shajani et al 2011).…”

60S ribosome biogenesis requires rotation of the 5S ribonucleoprotein particle

“…Newly synthesized rRNA folds co-transcriptionally and recruits r-proteins and assembly factors to form the early pre-ribosomal particles, which gradually evolve through a series of intermediates into mature ribosomes [5][6][7][8] . Along this pathway, the immature rRNA requires extensive modification, processing and structural remodelling 1 .…”

The ITS1-5.8S rRNA gene -ITS2 sequence variability during the divergence of sweet-grass species (gen us Glyceria R. Br.)

“…По подсчетам Альварец и Венделя (Alvarez, Wendel, 2003), сравнительное исследование района ITS1-5.8S рДНК-ITS2 проводилось в 66 % статей, касающихся вопросов ДНК-фи-логении растений. Несмотря на то, что биологические функции районов ITS до сих пор не вполне понятны (но они определенно играют важную роль в процессинге пре-рРНК и, возможно, некоторых иных РНК) (Calonje et al, 2009;Coleman, 2009;Koš, Tollervey, 2010), практика показывает, что филоге-нетические гипотезы, построенные на основании сравнения последователь-ностей ITS1 и ITS2, как правило, выглядят вполне правдоподобно, ведется ли сравнение на межвидовом и межродовом уровнях, или при изучении групп организмов, имеющих более высокие таксономические ранги (Nieto Feliner, Rossello, 2007;Шнеер, 2009;Coleman, 2009). Исследование ITS1 и ITS2 мо-жет позволить выяснить или ограничить круг предков объекта исследования не только по материнской линии родства, как это происходит при исследо-вании хлоропластных последовательностей, но, с некоторой вероятностью, и по отцовской линии.…”