Abstract
The synthesis of ribosomes involves the correct folding of the pre-ribosomal RNA within pre-ribosomal particles. The first ribosomal precursor or small subunit processome assembles stepwise on the nascent transcript of the 35S gene. At the earlier stages, the pre-ribosomal particles undergo structural and compositional changes, resulting in heterogeneous populations of particles with highly flexible regions. Structural probing methods are suitable for resolving these structures and providing evidence about the architecture of ribonucleoprotein complexes. Our approach used MNase tethered to the assembly factors Nan1/Utp17, Utp10, Utp12, and Utp13, which among other factors, initiate the formation of the small subunit processome. Our results provide dynamic information about the folding of the pre-ribosomes by elucidating the relative organization of the 5′ETS and ITS1 regions within the 35S and U3 snoRNA around the C-terminal domains of Nan1/Utp17, Utp10, Utp12, and Utp13.
Highlights
Ribosome biogenesis is an essential process for cell biology
We performed structural probing on affinity-purified preribosomal particles
Pre-ribosomal particles were affinity purified with an assembly factor (AF) fused to the TAP tag [30]
Summary
Ribosome biogenesis is an essential process for cell biology. In eukaryotic cells, the ribosomal RNAs (rRNAs) 18S, 5.8S, and 25S form part of a single pre-rRNA (35S), and they are separated by flanking sequences absent in the mature ribosomes (Figure 1A).These flanking sequences are removed during the maturation of the pre-ribosomal particles.More than 200 assembly factors (AFs) participate in the pre-ribosomal particles’ maturation, a process including the stable incorporation of ribosomal proteins, modification of nucleotides, removal of flanking sequences, and folding of the pre-rRNA.Several AFs associate with the nascent pre-rRNA to form the earliest identified preribosomal precursor [1,2,3]. The ribosomal RNAs (rRNAs) 18S, 5.8S, and 25S form part of a single pre-rRNA (35S), and they are separated by flanking sequences absent in the mature ribosomes (Figure 1A). Formation of the first precursor of the small ribosomal subunit (SSU) or SSU-processome is a stepwise process driven by the hierarchical relationships between different AFs and folding of the pre-rRNA [4,5,6,7]. In this regard, some AFs might function as platforms for the subsequent assembly of other AFs [8].
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