Deciphering the biogenesis of the Signal Recognition Particle, and its connections with ribosome biogenesis and nucleolar structure. – BioRiboSRP

Many cellular functions are achieved by a family of molecular machines made of RNA-protein complexes, called non-coding ribonucleoprotein particles (ncRNP). Understanding how these complexes are faithfully produced in cells and how the amount produced is controlled are essential issues. Many factors are dedicated to the biogenesis of ncRNPs. These factors chaperone free subunits, increase the specificity of assembly, control the quality of the particles produced, etc, … How ncRNP assembly is catalyzed and regulated is still poorly understood. This project is dedicated to the study of the biogenesis of the universally conserved Signal Recognition Particle (SRP), which is essential for targeting transmembrane and secretory proteins to the endoplasmic reticulum. Despite the essential role of SRP in cells, its mechanism of biogenesis is far from being understood. Remarkably, apart from the SMN complex, only very few factors involved in the biogenesis of mammalian SRP have been identified so far. SRP assembly takes place mainly in the nucleolus where most of the SRP proteins associate with the 7SL RNA, while SRP54 association occurs later in the cytoplasm.
The reason why SRP assembles in the nucleolus is not known. Since the nucleolus is the place where the initial steps of ribosome synthesis occur, SRP assembly in this compartment may benefit from the ribosome assembly factors enriched there. Since SRP and ribosomes are destined to work together in protein secretion through the co-translational recruitment of ribosomes at the ER, we entertain the possibility that their biogenesis may be coordinated. We already have bona fide evidence that this hypothesis is correct and we want to explore it further.

In this project, we will identify the pathway of SRP biogenesis and the trans-acting factors involved, using several complementary unbiased high-throughput approaches, including 1) quantitative proteomics to identify the SRP subcomplexes and assembly intermediates, as well as the proteins associated with each of the SRP proteins; and 2) large-scale screens with visual readouts to detect proteins whose absence leads to defects in SRP biogenesis. We will examine functionally in detail the most promising factors identified in our screens, using a battery of in cellulo and in vitro approaches. We will identify assembly intermediates by expressing mutants of SRP proteins or depleting each one of the SRP proteins. We will also identify the post-transcriptional modification landscape of 7SL RNA and which factors are involved. Overall, these experiments will provide a well-defined sequence of events leading to the formation of mature SRP, and identify common assembly factors between SRP and ribosome biogenesis.
The nucleolus is highly dynamic and is a biomarker of the health status in cells. Disrupting nucleolar functions and structure may impact SRP biogenesis and, reciprocally, efficient SRP biogenesis may be required to maintain nucleolar homeostasis. Thus, another goal of our project is to challenge this exciting novel concept using high-resolution microscopy.

Given the fundamental roles played by the SRP, it is essential to understand how it is assembled and how this assembly is regulated, and to characterize the trans-acting factors involved in its biogenesis. Moreover, by exploring the involvement of the nucleolus in SRP biogenesis, we anticipate unraveling novel insightful connections in the biogenesis of two essential cellular nanomachines: the ribosome and the SRP. Our consortium is composed of a powerful complementary combination of researchers, specialists in ncRNP biogenesis, and nucleolar structure and functions. This consortium is perfectly suited to allow this project to succeed, and to provide the community with important novel insights in RNA-protein macromolecular assembly, applicable to other types of machinery.