PRMT1-SFPQ regulates intron retention to control matrix gene expression during craniofacial development

Reviewer #1 (Public review):

The central finding of the current manuscript is that embryonic ablation of PRMT1 results in a craniofacial phenotype that is primarily linked to downstream intron splicing defects. This manuscript is one of several to underscore the relative importance of intron splicing to gene expression regulation during development, and moreover, to recapitulate splicing-related craniofacial defects. Specifically, authors introduce a regulatory axis consisting of PRMT1-SFPQ that directs mechanisms of long intron retention. This finding represents a significant contribution to our understanding of splicing regulation, in the sense that it highlights the regulatory impact that post-translational modification of splicing-related proteins can have on intron processing. Further, it emphasizes the importance of extending the study of splicing regulation beyond core components of the spliceosome, to include their upstream regulators as well.

The significance of neural crest cells in the development of craniofacial structures has long been considered a major contributor to developmental phenotypes. This specific symptomology is heavily associated with spliceosomopathies, wherein disruption of spliceosome components is the primary mechanism of disease pathogenesis. Thus, the PRMT1 associated phenotype is noteworthy. The role of PRMT1 in methylating downstream splicing factors introduces a new avenue of research focused on the mechanisms of spliceosome component activation and their effects on splicing. The strength of the current study lies in their establishing the molecular mechanism through which PRMT1 could alter craniofacial development through regulation of the transcriptome, but the data presented to support the claim that a PRMT1-SFPQ axis directly regulates intron retention of the relevant gene networks should be robust and with multiple forms of clear validation. For example, elevated intron retention findings are based on the intron retention index, and according to the manuscript, are assessed considering the relative expression of exons and introns from a given transcript. However, delineating between intron retention and other forms of alternative splicing (i.e., cryptic splice site recognition) requires a more comprehensive consideration of the intron splicing defects that could be represented in data. A certain threshold of intron read coverage (i.e., the percent of an intron that is covered by mapped reads) is needed to ascertain if those that are proximal to exons could represent alternative introns ends rather than full intron retention events. In other words, intron retention is a type of alternative splicing that can be difficult to analyze in isolation given the confounding influence of cryptic splicing and cryptic exon inclusion. If other forms of alternative splicing were assessed and not detected, more confident retention calls can be made.

While data presented to support the PRMT1-SFPQ activation axis is quite compelling, that this is directly responsible for the elevated intron retention remains enigmatic. First, in characterizing their PRMT1 knockout model, it is unclear whether the elevated intron retention events directly correspond to downregulated genes. Moreover, intron splicing is a well-documented node for gene regulation during embryogenesis and in other proliferation models, and craniofacial defects are known to be associated with 'spliceosomopathies'. However, reproduction of this phenotype does not suggest that the targets of interest are inherently splicing factors, and a more robust assessment is needed to determine the exact nature of alternative splicing in this system. Because there are several known splicing factors downstream of PRMT1 and presented in the supplemental data, the specific attribution of retention to SFPQ would be additionally served by separating its splicing footprint from that of other factors that are primed to cause alternative splicing.

Clarifying the relationship between SFPQ and splicing regulation is important given that the observed splicing defects are incongruous with published data presented by Takeuchi et al., (2018) regarding SFPQ control of neuronal apoptosis in mice. In this system, SFPQ was more specifically attributed to the regulation of transcription elongation over long introns and its knockout did not result in significant splicing changes. Thus, to establish the specificity for the SFPQ in regulating these retention events, authors would need to show that the same phenotype is not achieved by mis-regulation of other splicing factors. That the authors chose SFPQ based on its binding profile is understandable but potentially confounding given its mechanism of action in transcription of long introns (Takeuchi 2018). Because mechanisms and rates of transcription can influence splicing and exon definition interactions, the role of SFPQ as a transcription elongation factor versus a splicing factor is inadequately disentangled by authors.

Reviewer #2 (Public review):

Summary:
The manuscript by Lima et al examines the role of Prmt1 and SFPQ in craniofacial development. Specifically, the authors test the idea that Prmt1 directly methylates specific proteins that results in intron retention in matrix proteins. The protein SFPQ is methylated by Prmt1 and functions downstream to mediate Prmt1 activity. The genes with retained introns activate the NMD pathway to reduce the RNA levels. This paper describes an interesting mechanism for the regulation of RNA levels during development.

Strengths:
The phenotypes support what the authors claim that Prmt1 is involved in craniofacial development and splicing. The use of state-of-the-art sequencing to determine the specific genes that have intron retention and changes in gene expression is a strength.

Weaknesses:
Some of the data seems to contradict the conclusions. And it is unclear how direct the relationships are between Prmt1 and SFPQ.