The Small Nucle(ol)ar RNA Cap Trimethyltransferase Is Required for Ribosome Synthesis and Intact Nucleolar Morphology

Abstract

Nucleolar morphogenesis is a poorly defined process. Here we report that the Saccharomyces cerevisiae nucleolar trimethyl guanosine synthase I (Tgs1p), which specifically selects the m 7 G cap structure of snRNAs and snoRNAs for m 2,2,7 G conversion, is required not only for efficient pre-mRNA splicing but also for pre-rRNA processing and small ribosomal subunit synthesis. Mutational analysis indicates that the requirement for Tgs1p in pre-mRNA splicing, but not its involvement in ribosome synthesis, is dependent upon its function in cap trimethylation. In addition, we report that cells lacking Tgs1p showed a striking and unexpected loss of nucleolar structural organization. Tgs1p is not a core component of the snoRNP proteins; however, in vitro, the protein interacts with the KKD/E domain present at the carboxyl-terminal ends of several snoRNP proteins. Strains expressing versions of the snoRNPs lacking the KKD/E domain were also defective for nucleolar morphology and showed a loss of nucleolar compaction. We propose that the transient and functional interactions of Tgs1p with the abundant snoRNPs, through presumed interactions with the KKD/E domain of the snoRNP proteins, contribute substantially to the coalescence of nucleolar components. This conclusion is compatible with a model of self-organization for nucleolar assembly.The driving forces that govern the dynamic behavior of cellular organelles (i.e., their shape and size and their emergence at specific cellular foci) are unknown. The concept of self-organization that describes the ability of a macromolecular complex, or organelle, to determine its own structure on the basis of the functional and transient interactions of its constituents is particularly attractive (reviewed in reference 17). In a self-organized system, a steady-state structure is generated from highly dynamic components, i.e., "ever-changing partners," without the need for a rigid architectural framework. Classically, selforganization is opposed to self-assembly, where the physical associations of molecules generate a stable, static structure that reaches a "true equilibrium" (e.g., viruses and phages).Ribosome synthesis is a dynamic, multistep process that takes place largely in the nucleolus, a specialized region of the nucleus that is highly enriched in RNA-processing factors (RRPs) and further compartmentalized in individual domains, which accounts for the vectorial formation of the ribosomes (reviewed in references 21 and 26). The nucleolus appears as a steady-state compartment rather than a static structure. Indeed, during pre-ribosome assembly, pre-rRNAs, ribosomal proteins (RPs), and several hundred trans-acting factors, including the small nucleolar RNAs (snoRNAs), interact transiently (reviewed in references 2 and 10). Furthermore, major nucleolar antigens, such as fibrillarin and nucleolin are continuously exchanged with the surrounding nucleoplasm (1, 22; reviewed in reference 18).The spliceosomal RNAs U1, U2, U4, and U5, as well as several snoRNAs, are individually transcribe...

Mechanism of the AAA+ ATPases pontin and reptin in the biogenesis of H/ACA RNPs

“…5D). Interestingly, the charged tail of yeast NAP57, Cbf5p, is essential for cell viability (Jiang et al 1993) and that of yeast NOP56 and NOP58 is important for nucleolar integrity (Colau et al 2004). In summary, our model seems to apply to general snoRNP biogenesis.…”

General, rapid, and transcription-dependent fragmentation of nucleolar antigens in S. cerevisiae mRNA export mutants

“…Nucleolar morphology is most likely determined by the transient functional and physical interactions of its constituents, and seemingly small changes can have profound structural consequences; e.g., deficient capping of snoRNAs leads to loss of nucleolar organization (Colau et al 2004). The dramatic changes in molecular environment that occur in S. cerevisiae mRNA export mutants therefore reinforce the need for examining mRNA-related phenotypes at the earliest possible time point after transfer to restrictive conditions.…”

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

“…RNA extraction and Northern blotting were as described previously by Colau et al (2004). Oligonucleotides depicted in Supplemental Figure S1 are listed in Supplemental Table S6.…”