A Direct Interaction between the Utp6 Half-a-Tetratricopeptide Repeat Domain and a Specific Peptide in Utp21 Is Essential for Efficient Pre-rRNA Processing

Abstract

The small subunit (SSU) processome is a ribosome biogenesis intermediate that assembles from its subcomplexes onto the pre-18S rRNA with yet unknown order and structure. Here, we investigate the architecture of the UtpB subcomplex of the SSU processome, focusing on the interaction between the half-a-tetratricopeptide repeat (HAT) domain of Utp6 and a specific peptide in Utp21. We present a comprehensive map of the interactions within the UtpB subcomplex and further show that the N-terminal domain of Utp6 interacts with Utp18 while the HAT domain interacts with Utp21. Using a panel of point and deletion mutants of Utp6, we show that an intact HAT domain is essential for efficient pre-rRNA processing and cell growth. Further investigation of the Utp6-Utp21 interaction using both genetic and biophysical methods shows that the HAT domain binds a specific peptide ligand in Utp21, the first example of a HAT domain peptide ligand, with a dissociation constant of 10 M.In eukaryotes, ribosome biogenesis requires the coordinated processing and assembly of four ribosomal RNAs (rRNAs) and about 78 ribosomal proteins (49). In Saccharomyces cerevisiae, the 35S pre-rRNA is transcribed by RNA polymerase I in the nucleolus and is cleaved in several places to produce the mature 18S, 5.8S, and 25S rRNAs (Fig. 1). This process utilizes over 180 trans-acting factors and yet occurs fast enough to allow 2,000 ribosomes to be made each minute (for reviews, see references 16, 17, 22, 24, and 51). In this highly coordinated process, many factors assemble onto the nascent pre-rRNA as it is transcribed, forming large ribonucleoproteins (RNPs) that mature the small subunit (SSU) or large subunit (LSU) of the ribosome. Despite the identification of such trans-acting factors, the details of ribosome biogenesis remain elusive, as the complexity of ribosome biogenesis lies in the folding of prerRNAs and ribosomal proteins into functional ribosomes. Indeed, the frontier of ribosome synthesis investigation is to delineate the role(s) of each trans-acting factor in the production of the rRNA as it is modified, processed, and folded to become the mature ribosome (22).Ribosome biogenesis is a dynamic process in which transacting factors associate with and dissociate from the evolving pre-rRNA throughout its maturation. The initial assembly of factors involved in both SSU and LSU biogenesis with the 35S pre-rRNA has been termed the 90S preribosome, which is separated by cleavage in ITS1 into the SSU processome (required for SSU maturation) and the 66S preribosome (required for LSU maturation) (22). An RNP forming around the pre-18S rRNA has long been visualized in Miller chromatin spreads as a packed structure (35)

RNA binding and RNA remodeling activities of the half-a-tetratricopeptide (HAT) protein HCF107 underlie its effects on gene expression

“…Early reports speculated that HAT repeats might bind RNA (1,5,7). However, a lack of evidence for RNA binding and the fact that some HAT repeats can bind peptides (8,9) have led recent literature to assume that HAT motifs serve only to support protein-protein interactions (3, 9, 10). Our demonstration that the HAT protein HCF107 binds RNA with high affinity and sequence specificity together with the exclusive association of HAT proteins with RNA-containing complexes strongly suggest that RNA binding activity contributes to the biological functions of most HAT domains.…”

“…Crystal structures of CstF-77 confirmed that HAT repeat tracts adopt a TPR-like structure (3, 4). The possibility that HAT repeat tracts might bind RNA has been suggested (1, 5-7), but the notion that HAT repeat tracts serve as a scaffold for binding other proteins dominates recent literature (3,(8)(9)(10)). This view is reflected by the annotation of the HAT motif at InterPro, which states only that "the repeats may be involved in protein-protein interactions" (http://www.ebi.…”

The DEAD-box RNA helicase-like Utp25 is an SSU processome component

“…We therefore wished to determine if direct, binary protein-protein interactions form the basis of the co-complex interactions of Utp25 with Utp3 and Mpp10. Furthermore, while co-complex data are available for the t-Utp/UtpA, UtpB, and UtpC subcomplexes (Krogan et al 2004) and binary protein-protein interaction maps exist for the t-Utp/UtpA (Freed and Baserga 2010), UtpB (Champion et al 2008), and Mpp10 (Lee and Baserga 1999) subcomplexes, the interacting partners of the majority of SSU processome proteins remain undetermined. Therefore, we used a directed yeast two-hybrid approach to map the protein-protein interactions among Utp25 and Utp3 and their potential contacts with the UtpB and Mpp10 subcomplexes.…”

“…Some of its component proteins are known to associate into preformed subcomplexes, namely, the transcriptional Utps (t-Utps or UtpA subcomplex: t- Utp4,5,8,9,10,15,17,and Pol5) that link pre-rRNA transcription to processing Krogan et al 2004;Granneman and Baserga 2005), the UtpB subcomplex (Utp1, 6, 12, 13, 18, and 21) (Krogan et al 2004;Champion et al 2008), and the UtpC subcomplex (Utp22, Rrp7, and the four subunits of casein kinase II) (Krogan et al 2004). We have previously used the yeast two-hybrid methodology to map the protein-protein interactions within the UtpB subcomplex (Champion et al 2008), giving some insight into how the subcomplex is assembled from its individual components. Similarly, we have also shown that Mpp10 associates with Imp3 and Imp4 (Lee and Baserga 1999).…”

The nuclear poly(A) polymerase and Exosome cofactor Trf5 is recruited cotranscriptionally to nucleolar surveillance

“…UTP-A binds very early to nascent prerRNAs in a step that impinges on further SSU-processome components recruitment, including that of U3 itself (PerezFernandez et al 2007). It is not yet known whether UTP-B performs a specific function (Champion et al 2008). UTP-C integrates pre-rRNA processing and r-protein genes transcription (Rudra et al 2007).…”