Srsf3 mediates alternative RNA splicing downstream of PDGFRα signaling

Signaling through the platelet-derived growth factor receptor alpha (PDGFRα) is critical for mammalian craniofacial development, though the mechanisms by which the activity of downstream intracellular effectors is regulated to mediate gene expression changes have not been defined. We find that the RNA-binding protein Srsf3 is phosphorylated at Akt consensus sites downstream of PI3K-mediated PDGFRα signaling in palatal mesenchyme cells, leading to its nuclear translocation. We further demonstrate that ablation of Srsf3 in the neural crest lineage leads to facial clefting due to defective cranial neural crest cell specification and survival. Finally, we show that Srsf3 regulates the alternative RNA splicing of transcripts encoding protein kinases in the facial process mesenchyme to negatively regulate PDGFRα signaling. Collectively, our findings reveal that PI3K/Akt-mediated PDGFRα signaling primarily modulates gene expression through alternative RNA splicing in the facial mesenchyme and identify Srsf3 as a critical regulator of craniofacial development.

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Srsf3 mediates alternative RNA splicing downstream of PDGFRα signaling in the facial mesenchyme

Development ◽

10.1242/dev.199448 ◽

2021 ◽

Author(s):

Brenna J.C. Dennison ◽

Eric D. Larson ◽

Rui Fu ◽

Julia Mo ◽

Katherine A. Fantauzzo

Keyword(s):

Gene Expression ◽

Neural Crest ◽

Intracellular Signaling ◽

Rna Splicing ◽

Rna Binding ◽

Gene Expression Regulation ◽

Nuclear Translocation ◽

Growth Factor Receptor ◽

Neural Crest Cell ◽

Craniofacial Development

Signaling through the platelet-derived growth factor receptor alpha (PDGFRα) is critical for mammalian craniofacial development, though the mechanisms by which the activity of downstream intracellular effectors is regulated to mediate gene expression changes have not been defined. We find that the RNA-binding protein Srsf3 is phosphorylated at Akt consensus sites downstream of PI3K-mediated PDGFRα signaling in mouse palatal mesenchyme cells, leading to its nuclear translocation. We further demonstrate that ablation of Srsf3 in the mouse neural crest lineage leads to facial clefting due to defective cranial neural crest cell proliferation and survival. Finally, we show that Srsf3 regulates the alternative RNA splicing of transcripts encoding protein kinases in the mouse facial process mesenchyme to regulate PDGFRα-dependent intracellular signaling. Collectively, our findings reveal that alternative RNA splicing is an important mechanism of gene expression regulation downstream of PI3K/Akt-mediated PDGFRα signaling in the facial mesenchyme and identify Srsf3 as a critical regulator of craniofacial development.

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The Role of RNA-Binding Proteins in Vertebrate Neural Crest and Craniofacial Development

Journal of Developmental Biology ◽

10.3390/jdb9030034 ◽

2021 ◽

Vol 9 (3) ◽

pp. 34

Author(s):

Thomas E. Forman ◽

Brenna J. C. Dennison ◽

Katherine A. Fantauzzo

Keyword(s):

Gene Expression ◽

Neural Crest ◽

Binding Proteins ◽

Rna Binding ◽

Gene Expression Regulation ◽

Rna Binding Proteins ◽

Craniofacial Development ◽

Specific Sequence ◽

Altered Expression ◽

Binding Domains

Cranial neural crest (NC) cells delaminate from the neural folds in the forebrain to the hindbrain during mammalian embryogenesis and migrate into the frontonasal prominence and pharyngeal arches. These cells generate the bone and cartilage of the frontonasal skeleton, among other diverse derivatives. RNA-binding proteins (RBPs) have emerged as critical regulators of NC and craniofacial development in mammals. Conventional RBPs bind to specific sequence and/or structural motifs in a target RNA via one or more RNA-binding domains to regulate multiple aspects of RNA metabolism and ultimately affect gene expression. In this review, we discuss the roles of RBPs other than core spliceosome components during human and mouse NC and craniofacial development. Where applicable, we review data on these same RBPs from additional vertebrate species, including chicken, Xenopus and zebrafish models. Knockdown or ablation of several RBPs discussed here results in altered expression of transcripts encoding components of developmental signaling pathways, as well as reduced cell proliferation and/or increased cell death, indicating that these are common mechanisms contributing to the observed phenotypes. The study of these proteins offers a relatively untapped opportunity to provide significant insight into the mechanisms underlying gene expression regulation during craniofacial morphogenesis.

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Pdgfra and Pdgfrb genetically interact in the murine neural crest cell lineage to regulate migration and proliferation

10.1101/2020.07.29.227306 ◽

2020 ◽

Author(s):

Julia Mo ◽

Robert Long ◽

Katherine A. Fantauzzo

Keyword(s):

Neural Crest ◽

Tyrosine Kinases ◽

Genetic Interaction ◽

Cell Lineage ◽

Growth Factor Receptor ◽

Neural Crest Cell ◽

Craniofacial Development ◽

Late Gestation ◽

Allelic Series ◽

Cellular Mechanisms

AbstractCranial neural crest cells (cNCCs) are migratory, multipotent cells that originate from the forebrain to the hindbrain and eventually give rise to the bone and cartilage of the frontonasal skeleton, among other derivatives. Signaling through the two members of the platelet-derived growth factor receptor (PDGFR) family of receptor tyrosine kinases, alpha and beta, plays critical roles in the cNCC lineage to regulate craniofacial development during murine embryogenesis. Further, the PDGFRs have been shown to genetically interact during murine craniofacial development at mid-to-late gestation. Here, we examined the effect of ablating both Pdgfra and Pdgfrb in the murine NCC lineage on earlier craniofacial development and determined the cellular mechanisms by which the observed phenotypes arose. Our results confirm a genetic interaction between the two receptors in this lineage, as phenotypes observed in an allelic series of mutant embryos often worsened with the addition of conditional alleles. The defects observed here were shown to stem from reduced cNCC stream size and aberrant cNCC directional migration, as well as decreased proliferation of the facial mesenchyme upon combined decreases in PDGFRα and PDGFRβ signaling. Importantly, we found that PDGFRα plays a predominant role in cNCC migration whereas PDGFRβ primarily contributes to proliferation of the facial mesenchyme. Our findings provide insight into the distinct mechanisms by which PDGFRα and PDGFRβ signaling regulate cNCC activity and subsequent craniofacial development in the mouse embryo.

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Vgll2a is required for neural crest cell survival during zebrafish craniofacial development

Developmental Biology ◽

10.1016/j.ydbio.2011.06.034 ◽

2011 ◽

Vol 357 (1) ◽

pp. 269-281 ◽

Cited By ~ 27

Author(s):

Christopher W. Johnson ◽

Laura Hernandez-Lagunas ◽

Weiguo Feng ◽

Vida Senkus Melvin ◽

Trevor Williams ◽

...

Keyword(s):

Neural Crest ◽

Cell Survival ◽

Neural Crest Cell ◽

Craniofacial Development ◽

Crest Cell

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16p12.1 deletion orthologs are expressed in motile neural crest cells and are important for regulating craniofacial development in Xenopus laevis

10.1101/2020.12.11.421347 ◽

2020 ◽

Author(s):

Micaela Lasser ◽

Jessica Bolduc ◽

Luke Murphy ◽

Caroline O'Brien ◽

Sangmook Lee ◽

...

Keyword(s):

Xenopus Laevis ◽

Neural Crest ◽

Expression Patterns ◽

Copy Number Variants ◽

Neural Crest Cell ◽

Underlying Mechanism ◽

Craniofacial Development ◽

Pharyngeal Arches ◽

Individual Gene ◽

Vertebrate Model

Copy number variants (CNVs) associated with neurodevelopmental disorders are characterized by extensive phenotypic heterogeneity. In particular, one CNV was identified in a subset of children clinically diagnosed with intellectual disabilities (ID) that results in a hemizygous deletion of multiple genes at chromosome 16p12.1. In addition to ID, individuals with this deletion display a variety of symptoms including microcephaly, seizures, cardiac defects, and growth retardation. Moreover, patients also manifest severe craniofacial abnormalities, such as micrognathia, cartilage malformation of the ears and nose, and facial asymmetries; however, the function of the genes within the 16p12.1 region have not been studied in the context of vertebrate craniofacial development. The craniofacial tissues affected in patients with this deletion all derive from the same embryonic precursor, the cranial neural crest, leading to the hypothesis that one or more of the 16p12.1 genes may be involved in regulating neural crest cell (NCC)-related processes. To examine this, we characterized the developmental role of the 16p12.1-affected gene orthologs, polr3e, mosmo, uqcrc2, and cdr2, during craniofacial morphogenesis in the vertebrate model system, Xenopus laevis. While the currently-known cellular functions of these genes are diverse, we find that they share similar expression patterns along the neural tube, pharyngeal arches, and later craniofacial structures. As these genes show co-expression in the pharyngeal arches where NCCs reside, we sought to elucidate the effect of individual gene depletion on craniofacial development and NCC migration. We find that reduction of several 16p12.1 genes significantly disrupts craniofacial and cartilage formation, pharyngeal arch migration, as well as NCC specification and motility. Thus, we have determined that some of these genes play an essential role during vertebrate craniofacial patterning by regulating specific processes during NCC development, which may be an underlying mechanism contributing to the craniofacial defects associated with the 16p12.1 deletion.

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Nucleo-cytoplasmic shuttling of splicing factor SRSF1 is required for development and cilia function

10.1101/2020.09.04.263251 ◽

2020 ◽

Cited By ~ 2

Author(s):

Fiona Haward ◽

Magdalena M. Maslon ◽

Patricia L. Yeyati ◽

Nicolas Bellora ◽

Jan N. Hansen ◽

...

Keyword(s):

Gene Expression ◽

Mouse Model ◽

Rna Splicing ◽

Rna Binding ◽

Rna Binding Proteins ◽

Small Body ◽

Splicing Factor ◽

Nuclear Retention ◽

Ciliary Ultrastructure ◽

Retention Signal

AbstractShuttling RNA-binding proteins coordinate nuclear and cytoplasmic steps of gene expression. The SR family proteins regulate RNA splicing in the nucleus and a subset of them, including SRSF1, shuttles between the nucleus and cytoplasm affecting post-splicing processes. However, the physiological significance of this remains unclear. Here, we used genome editing to knock-in a nuclear retention signal (NRS) in Srsf1 to create a mouse model harboring an SRSF1 protein that is retained exclusively in the nucleus. Srsf1NRS/NRS mutants displayed small body size, hydrocephalus and immotile sperm, all traits associated with ciliary defects. We observed reduced translation of a subset of mRNAs and decreased abundance of proteins involved in multiciliogenesis, with disruption of ciliary ultrastructure and motility in cells derived from this mouse model. These results demonstrate that SRSF1 shuttling is used to reprogram gene expression networks in the context of high cellular demands, as observed here, during motile ciliogenesis.

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Protein-RNA interactome analysis reveals wide association of KSHV ORF57 with host non-coding RNAs and polysomes

Journal of Virology ◽

10.1128/jvi.01782-21 ◽

2021 ◽

Author(s):

Beatriz Alvarado-Hernandez ◽

Yanping Ma ◽

Nishi R. Sharma ◽

Vladimir Majerciak ◽

Alexei Lobanov ◽

...

Keyword(s):

Gene Expression ◽

Rna Splicing ◽

Rna Binding ◽

Viral Gene Expression ◽

Viral Gene ◽

28S Rrna ◽

Protein Coding ◽

Infected Cells ◽

Non Coding Rnas ◽

Rna Interaction

Kaposi’s sarcoma-associated herpesvirus (KSHV) ORF57 is an RNA-binding post-transcriptional regulator. We recently applied an affinity-purified anti-ORF57 antibody to conduct ORF57-CLIP (Cross-linking Immunoprecipitation) in combination with RNA-sequencing (CLIP-seq) and analyzed the genome-wide host RNA transcripts in association with ORF57 in BCBL-1 cells with lytic KSHV infection. Mapping of the CLIPed RNA reads to the human genome (GRCh37) revealed that most of the ORF57-associated RNA reads were from rRNAs. The remaining RNA reads mapped to several classes of host non-coding and protein-coding mRNAs. We found ORF57 binds and regulates expression of a subset of host lncRNAs, including LINC00324, LINC00355, and LINC00839 which are involved in cell growth. ORF57 binds snoRNAs responsible for 18S and 28S rRNA modifications, but does not interact with fibrillarin and NOP58. We validated ORF57 interactions with 67 snoRNAs by ORF57-RNA immunoprecipitation (RIP)-snoRNA-array assays. Most of the identified ORF57 rRNA binding sites (BS) overlap with the sites binding snoRNAs. We confirmed ORF57-snoRA71B RNA interaction in BCBL-1 cells by ORF57-RIP and Northern blot analyses using a 32 P-labeled oligo probe from the 18S rRNA region complementary to snoRA71B. Using RNA oligos from the rRNA regions that ORF57 binds for oligo pulldown-Western blot assays, we selectively verified ORF57 interactions with 5.8S and 18S rRNAs. Polysome profiling revealed that ORF57 associates with both monosomes and polysomes and its association with polysomes increases PABPC1 binding to, but prevent Ago2 from polysomes. Our data indicate a functional correlation with ORF57 binding and suppression of Ago2 activities for ORF57 promotion of gene expression. Significance As an RNA-binding protein, KSHV ORF57 regulates RNA splicing, stability, and translation and inhibits host innate immunity by blocking the formation of RNA granules in virus infected cells. In this report, ORF57 was found to interact many host non-coding RNAs, including lncRNAs, snoRNAs and ribosomal RNAs to carry out additional unknown functions. ORF57 binds a group of lncRNAs via the identified RNA motifs by ORF57 CLIP-seq to regulate their expression. ORF57 associates with snoRNAs independently of fibrillarin and NOP58 proteins, and with ribosomal RNA in the regions that commonly bind snoRNAs. Knockdown of fibrillarin expression decreases the expression of snoRNAs and CDK4, but not affect viral gene expression. More importantly, we found that ORF57 binds translationally active polysomes and enhances PABPC-1 but prevents Ago2 association with polysomes. Data provide a compelling evidence on how ORF57 in KSHV infected cells might regulate protein synthesis by blocking Ago2’s hostile activities on translation.

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