Ribosomes are universally conserved macromolecular machines that translate cellular genetic information into proteins. All ribosomes are com- posed of two ribonucleoprotein subunits. In eukaryotes these are called 40S (small) and 60S (large) subunits. Biogenesis of both subunits begins from a common precursor ribosomal RNA (rRNA) transcript in the nucleolus. The 18S rRNA of the small subunit is encoded in the 5ʹ end of the precursor transcript. U3 snoRNA and about 70 accessory factors associate with the 50 end of the pre-rRNA, to form the SSU processome or 90S pre-ribosome, which can be observed as terminal knobs in electron micrographs. After the initial processing and folding, the pre-rRNA is cleaved at site A2 to release the pre--40S. This event is dependent on the formation of the central pseudoknot, a structure that maintains the integrity of 40S architecture. Bud23 is the methyltransferase responsible for modification of the base G1575 in the P-site of the small subunit. Work presented here demonstrates that the in vivo stability, and thus function, of Bud23 is dependent on the presence of Trm112, a novel ribosome biogenesis factor identified in this work. Analysis of rRNA processing and strong negative genetic interactions with RNaseMRP mutants, provide strong evidence for that BUD23 is required for A2 cleavage. Extragenic suppressors of bud23 [delta] were identified in UTP14, UTP2, IMP4 and ECM16, coding for SSU processome components. Bud23 and the RNA helicase Ecm16 interact physically as well as genetically. Most fascinatingly, using ecm16 enzymatic mutants, this work provides compelling evidence that Ecm16 facilitates removal of U3 snoRNA from pre-rRNA, a prerequisite for central pseudoknot formation and 90S to pre--40S transition. These findings suggest a model in which binding of Bud23 monitors the status of 40S assembly, triggering Ecm16 activity to promote release of the pre--40S from 90S only after the critical folding of the small subunit rRNA. / text

The ribosome is one of the most universal molecular machinery, synthesizing proteins in all living systems. The small ribosomal subunit plays a crucial role in decoding the messenger RNA during translation. We propose and validate a new architectural model of the ribosomal small subunit, with broad implications for function, biogenesis and evolution. We define an rRNA domain: compact and modular, stabilized by self-consistent molecular interactions, with ability to fold autonomously when it is isolated from surrounding RNA or protein. Each rRNA helix must be allocated uniquely to a single domain. These criteria identify a core domain of small subunit rRNA (domain A), which acts as a hub, linking to all other domains by A-form helical spokes. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2’OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with autonomous folding, and therefore classification as a domain. We show that the domain concept is applicable and useful for understanding the small ribosomal subunit. Our results support the utility of the concept of the domain as applied to at least some RNAs, the interdependence of the elements of domain A, and its ability to fold autonomously. Moreover, domain A, which exhibits elements of tRNA mimicry, is the essential core of the small ribosomal subunit. Understanding the structure and dynamics of domain A will provide valuable insight into the translational machinery.