Abstract
Ribosome biogenesis is a dynamic multistep process, many features of which are still incompletely documented. Here, we show that changes in this pathway can be captured and annotated by means of a graphic set of pre-rRNA ratios, a technique we call Ratio Analysis of Multiple Precursors (RAMP). We find that knocking down a ribosome synthesis factor produces a characteristic RAMP profile that exhibits consistency across a range of depletion levels. This facilitates the inference of affected steps and simplifies comparative analysis. We applied RAMP to examine how endonucleolytic cleavages of the mouse pre-rRNA transcript in the internal transcribed spacer 1 (ITS1) are affected by depletion of factors required for maturation of the small ribosomal subunit (Rcl1, Fcf1/Utp24, Utp23) and the large subunit (Pes1, Nog1). The data suggest that completion of early maturation in a subunit triggers its release from the common pre-rRNA transcript by stimulating cleavage at the proximal site in ITS1. We also find that splitting of pre-rRNA in the 3′ region of ITS1 is prevalent in adult mouse tissues and quiescent cells, as it is in human cells. We propose a model for subunit separation during mammalian ribosome synthesis and discuss its implications for understanding pre-rRNA processing pathways.
Highlights
IntroductionThe eukaryotic ribosome is made of four ribosomal RNAs (rRNAs) and around 80 ribosomal proteins
The eukaryotic ribosome is made of four ribosomal RNAs and around 80 ribosomal proteins
Reliable identification of the ribosomal RNAs (rRNAs) maturation steps affected by deficiency of a ribosome assembly factor can be non-trivial, given that altered steady-state levels of prerRNAs often reflect changes in multiple steps of the pathway and vary depending on the extent of the factor’s depletion
Summary
The eukaryotic ribosome is made of four ribosomal RNAs (rRNAs) and around 80 ribosomal proteins. There are typically four spacers designated 5 ETS, ITS1, ITS2 and 3 ETS, which are removed from the pre-rRNA transcript in the course of its posttranscriptional maturation [1]. An important step in pre-rRNA processing is the endonucleolytic separation of the primary transcript within ITS1 (Figure 1A). The two parts of the transcript continue their maturation in a largely independent manner to form 18S rRNA in the small subunit (SSU) and 5.8S, 28S in the large subunit (LSU). Processing of pre-rRNA and assembly of the nascent subunits requires >200 transiently associating assembly factors that include enzymes (such as RNA helicases and ribonucleases) and proteins with non-enzymatic functions [2,3]
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