The SON RNA splicing factor is required for intracellular trafficking that promotes centriole assembly

  1. Alexander J. Stemm-Wolf
  2. Eileen T. O’Toole
  3. Ryan M. Sheridan
  4. Jacob T. Morgan
  5. Chad G. Pearson

  • Curated by eLife

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    Evaluation Summary:

    This study investigates how deficiency in the RNA splicing factor SON impairs centriole assembly, which may underlie ciliopathy-like phenotypes in humans with SON mutations and is thus of interest to both cell biologists and clinicians. Using RNA-sequencing analysis and advanced imaging techniques the authors discover a large number of known and new SON splicing targets and attempt to identify those crucial for SON knockdown defects. However, knockdown of a subset of targets did not fully recapitulate SON depletion phenotypes and only led to the relatively vague conclusion that the observed centriole assembly defects were caused by impaired protein trafficking around the centrosome.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

Control of centrosome assembly is critical for cell division, intracellular trafficking and cilia. Regulation of centrosome number occurs through the precise duplication of centrioles that reside in centrosomes. Here we explored transcriptional control of centriole assembly and find that the RNA splicing factor SON is specifically required for completing procentriole assembly. Whole genome mRNA sequencing identified genes whose splicing and expression are affected by the reduction of SON, with an enrichment in genes involved in the microtubule cytoskeleton, centrosome and centriolar satellites. SON is required for the proper splicing and expression of CEP131 which encodes a major centriolar satellite protein and is required to organize the trafficking and microtubule network around the centrosomes. This study highlights the importance of the distinct microtubule trafficking network that is intimately associated with nascent centrioles and is responsible for procentriole development.

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  1. eLife

    Evaluation Summary:

    This study investigates how deficiency in the RNA splicing factor SON impairs centriole assembly, which may underlie ciliopathy-like phenotypes in humans with SON mutations and is thus of interest to both cell biologists and clinicians. Using RNA-sequencing analysis and advanced imaging techniques the authors discover a large number of known and new SON splicing targets and attempt to identify those crucial for SON knockdown defects. However, knockdown of a subset of targets did not fully recapitulate SON depletion phenotypes and only led to the relatively vague conclusion that the observed centriole assembly defects were caused by impaired protein trafficking around the centrosome.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  2. eLife

    Reviewer #1 (Public Review):

    The manuscript by Stemm-Wolf et al. investigates the role of the splicing factor SON in centriole assembly, which was previously identified by others in a genome-wide screen for factors required for centriole duplication. The authors first confirm that SON impairs centriole duplication. They show that procentriole formation occurs, but elongation and proper centriolar microtubule triplet formation fail. Using mRNA sequencing, the authors extend the list of previously described SON splicing targets, providing the most comprehensive list to date, including many components of centrioles and centriolar satellites. The authors then attempt to identify targets crucial for SON's role in centriole assembly and found that depletion of CEP131, a centriolar satellite component, phenocopies SON depletion to some degree. They further observed alterations in centrosomal microtubule organization and a reduction in centrosome proteins in the vicinity of centrosomes after SON depletion, which makes them conclude that defective trafficking of centrosome components, as part of satellites or similar trafficking particles, is the main cause of the centriole assembly defects. However, altered centrosomal microtubule organization after SON knockdown was observed previously and there is very little data that goes beyond this finding. It also seems that, rather than conducting a more comprehensive analysis, the authors have focused on a few candidates that they thought would help to explain SON depletion phenotypes. However, those that have been tested, alone or in combination, do not or only to a limited extent recapitulate SON depletion and none was tested for an involvement in the altered centrosomal microtubule organization phenotype. Moreover, considering the long list of centrosome proteins affected by SON, the phenotype may not involve only one or two proteins. At the end the authors argue that katanin, also a splicing target of SON, may be involved in altered centrosomal microtubule organization, but there is no experiment to test this. In conclusion, despite various detailed analyses, there is not a major advance regarding mechanistic insight and the offered explanation that impaired trafficking around centrosomes causes centriole assembly defects after SON depletion is relatively vague.

  3. eLife

    Reviewer #2 (Public Review):

    Centriole number is tightly controlled in cells and deregulation of these controls cause various human diseases. In this manuscript, the authors investigated the transcriptional control of centriole assembly by focusing on characterization of the RNA splicing factor SON. To this end, they combined advanced microscopy approaches with functional assays and whole genome sequencing. They first showed that SON is required for canonical duplication and PLK4-induced centriole amplification. Through characterization of centrosomal levels of duplication factors in SON-depleted cells, they found that centriole assembly is initiated properly but not completed. Using EM, they reported structural defects in nascent centrioles. Additionally, they used whole genome RNA sequencing to identify genes that require SON for expression and splicing, which included a wide range of centriolar satellite proteins. By focusing on Cep131, PCM1, pericentrin, centrobin and katanin, they described defects in centrosomal recruitment and pericentrosomal clustering of these proteins, which in part explains how SON regulates centriole assembly. Collectively, they propose that SON regulates microtubule organization and centriole assembly by regulating trafficking of regulators of centriole assembly and microtubule dynamics.

    The results of the manuscript contribute to our understanding of transcript-level regulation of centriole biogenesis and microtubule organization. SON was previously described for its functions in centriole duplication, cell cycle progression and splicing of gamma-tubulin and pericentrin. Therefore, its functions as a regulator of centriole assembly is not unexpected. The authors extensively investigated the molecular mechanisms underlying these defects, which is challenging due to regulatory roles of SON on a wide range of centrosome/satellite/microtubule-associatd proteins as revealed by whole genome sequencing. To this end, they focused on key regulators of centrosome biogenesis.

    Although the authors suggest regulation of PCM1, pericentrin, centrobin, Cep131 and katanin and satellite-mediated trafficking as potential mechanisms by which mediates its centriolar functions, the data presented does not provide strong evidence for these conclusions. For example, Cep131 expression does not rescue the phenotypes in SON-depleted cells. Rescue experiments with other proteins have not been performed, which are essential to show a direct link between SON functions and its targets. Additionally, there is no data that supports the role of SON in intracellular trafficking around the centrosome (i.e. dynamic imaging of Cep131/pericentrin granules). Therefore, although the findings of the manuscript is of general interest and has potential to advance our understanding of transcriptional control of centrosome biogenesis, further experiments are required to justify their conclusions.

  4. eLife

    Reviewer #3 (Public Review):

    This manuscript by Stemm-Wolf and colleagues examines the role of the SON RNA-splicing factor in the transcriptional regulation of centriole assembly and centrosome protein composition. The authors use a combination of siRNA-mediated gene silencing, RNA sequencing/in silico analyses, super-resolution microcopy and electron tomography to investigate how loss of SON function impacts centriole duplication, centrosome protein composition, satellite-mediated protein trafficking and microtubule organization in cultured cells in vitro. The authors demonstrate that although depletion of SON is dispensable for early steps of procentriole assembly, loss of SON causes a bock in the completion of centriologenesis. Using RNA sequencing of SON-depleted cells, they identify a role for SON in splicing of genes encoding components of centriolar satellites, as well as a number of microtubule and centrosome associated proteins. Using immunofluorescence imaging, they confirm that SON loss alters the distribution of centrosomal and centriolar satellite proteins in cells, as well as microtubule nucleation at centrosomes.

    Overall, the manuscript is well written and the data are well presented. The strengths of the study are the use of advanced imaging techniques to visualize and quantify centrioles and centrosomes upon SON manipulation, and the RNA sequencing data that points to roles for SON in these processes. However, what remains unclear is the connection between SON-related splicing defects and the mechanisms that result in the observed centriole assembly defects. Loss of SON appears to impact a large number of genes (over 4000). With regards to centrosome biology, it appears to impact a variety of different centriolar, PCM, and satellite proteins, all of which could somehow cause these phenotypes. Thus, it was difficult for this reviewer to understand how exactly loss of SON causes dysregulation of procentriole growth, other than concluding that it impacts the function of numerous genes/proteins simultaneously. As the authors themselves note "the mechanism of SON-based regulation of centriole assembly is multifactorial". Although this makes sense to a certain degree, the same might be said of a number of splicing factors that control expression/splicing of large sets of genes. As such, the only conclusion I left with was that defects in gene splicing of centriolar/centrosomal genes can disrupt procentriole growth, which is not that surprising considering mis-splicing of even a single gene essential for procentriole assembly would result in this phenotype.

  5. Version published to 10.1101/2021.01.29.428802 on bioRxiv