Facilitated assembly of RNA pol III RNPs - biogenesis of the human SRP complex

The function of all cells requires that individual activities are harnessed and controlled, often within large molecular machines. Examples include the machineries that replicate DNA, synthesize proteins and generate energy (ATP). These complexes must be assembled correctly in a controlled way, far from where they function, as partially or incorrectly assembled complexes might interfere with the normal processes. We wish to study the assembly of one such cellular machine, the signal recognition particle that is intimately associated with protein secretion from cells. Previous understanding indicates that this takes place within the nucleus, and largely within a subnuclear region termed the nucleolus, which is also the site of assembly of a number of other complexes, notably the protein synthesizing ribosome. Our aim is to identify components of the assembly and localisation path of signal recognition particle and to determine the role that each of these plays in these events.

Complexes of non-coding RNA and protein (RNPs) play key roles in essential cellular processes. These include maintenance of chromatin ends (telomerase), dosage compensation, transcription, pre-mRNA splicing, translation and RNA turnover. Functionally important RNPs assembled on transcripts produced by RNA pol III (termed pol III RNPs) include U6 snRNP (splicing), 5S (translation), SRP (protein targeting), 7SK (transcription control), RNAses P (tRNA processing) and MRP (rRNA processing, mitochondrial DNA synthesis), Ro (stress response) and Vault (multi-drug resistance). Recent work has highlighted surprising complexity in RNP biogenesis. An emerging theme is that RNP assembly and localisation are facilitated and that the RNA/RNP undergoes quality control. Despite their key roles in basic cell biological processes, the biogenesis of pol III RNPs has received relatively little attention compared to pol I and II RNPs such as the ribosome and snRNPs. We wish to understand these events, and here we set out to study the biogenesis of human SRP as a model for pol III RNPs in general. SRP is an abundant cytoplasmic RNP that catalyses co-translational protein targeting to the endoplasmic reticulum membrane. SRP biogenesis has been linked to the nucleolus and SRP RNA is found in the peri-nucleolar compartment. Trafficking of the maturing complex to the cytoplasm implies that nuclear material represents pre-SRP complexes. We have already defined a novel role for nucleolin and confirmed the involvement of La and Lsm proteins in SRP biogenesis. Gradient fractionation revealed pre-SRP complexes 4 to 5 times larger that the mature particle implying the involvement of significantly more factors than previously assigned to this process. Identifying these additional proteins will provide a major breakthrough in understanding the biogenesis of SRP, and likely other pol III RNPs. Knowledge of the dynamics of protein association and dissociation with the pre-SRP will be fundamental to understanding this process. The work will use a combined in vitro biochemical and RNAi-based in vivo approach to identify pre-SRP associated factors, define their temporal and spatial association with the complex, and characterise their roles in SRP biogenesis.