A summary of the proposed interactions between mouse U3 snoRNA and pre-rRNA. Two regions (5′ hinge and 3′ hinge) in U3 snoRNA can base-pair with two sequences in the 5′ETS. Box A and A′ in U3 exhibit conserved complementarity to the 5′ region in 18S rRNA. According to the model of Borovjagin and Gerbi (2005), base-pairing between the U3 hinges and 5′ETS may be required for the spatial reorganization of the complex allowing binding of boxes A and A′ to the 18S sequences in pre-rRNA.

Analysis of the 5′ and 3′ ends formed by the novel cleavage in mouse 5′ETS. (A) Primer extension analysis with primer A0prEx4 to map the novel cleavage site (lane 1) and A′prEx2 to map the primary cleavage site (lane 2) are shown next to the matching sequencing reactions. The nucleotide sequence shown with the 5′ end at the top is colinear with pre-rRNA; major and minor polymerase stops are indicated by the large and small arrowheads, respectively. (B) 3′ ends of RNA fragments derived by processing at nucleotide 1673/1674 in EXOSC10-depleted cells. See Materials and Methods for details. Nontemplated bases are shown in italics. Numbering in pre-rRNA corresponds to the GenBank sequence X82564.

A revised map of pre-rRNA processing leading to 18S rRNA formation in mouse cells. The major parts on the primary transcript (47S) and cleavage sites are indicated at the top. The 47S pre-rRNA is normally short-lived in cells due to the rapidly occurring processing at sites A′ in 5′ETS and 6 in 3′ETS that generate 45S pre-rRNA. Subsequent cleavages may first occur either in ITS1 (probably at site 2c) or in 5′ETS (site A0). The end result of both pathways is the removal of the remaining portion of 5′ETS yielding 20S pre-rRNA, which is converted to 18S-E and finally processed to the mature 18S rRNA in the cytoplasm. Conventional names for the precursors are based on the nomenclature of Bowman et al. (1981) and Eichler and Craig (1994).

Depletion of EXOSC10 unmasks a second processing site in the 5′ETS of mouse pre-rRNA. (A) Structure of the mouse 47S pre-rRNA transcript showing major cleavage sites and relative position of hybridization probes. Novel pre-rRNAs generated by cleavage at the second site described here (indicated by an arrow) are shown in italics. (B) EXOSC10 protein level is reduced after transfection with specific siRNA, but not after transfections with a heterologous siRNA as compared with mock-transfected cells. Immunoblotting of cell lysates was perfomed with antibodies against PM-Scl100; beta-tubulin was used as a loading control. (C) Hybridization analysis of pre-rRNA. Total RNA was isolated from siControl-transfected (lanes 1,3,5,7,9,10) or siEXOSC10-transfected cells (lanes 2,4,6,8), separated by formaldehyde-agarose gel electrophoresis, blotted on a membrane, stained with methylene blue to reveal 28S, 18S rRNAs, and marker bands, and hybridized with the indicated oligonucleotide probes. Arrow points to the spacer fragment accumulating in EXOSC10-depleted cells. Lanes 9 and 10 show a close-up view of hybridizations in which the gel was run for a longer time to improve separation between 43S and 47/45S pre-rRNAs.