PLRG1 Is an Essential Regulator of Cell Proliferation and Apoptosis during Vertebrate Development and Tissue Homeostasis

Abstract

PLRG1, an evolutionarily conserved component of the spliceosome, forms a complex with Pso4/SNEV/Prp19 and the cell division and cycle 5 homolog (CDC5L) that is involved in both pre-mRNA splicing and DNA repair. Here, we show that the inactivation of PLRG1 in mice results in embryonic lethality at 1.5 days postfertilization. Studies of heart-and neuron-specific PLRG1 knockout mice further reveal an essential role of PLRG1 in adult tissue homeostasis and the suppression of apoptosis. PLRG1-deficient mouse embryonic fibroblasts (MEFs) fail to progress through S phase upon serum stimulation and exhibit increased rates of apoptosis. PLRG1 deficiency causes enhanced p53 phosphorylation and stabilization in the presence of increased ␥-H2AX immunoreactivity as an indicator of an activated DNA damage response. p53 downregulation rescues lethality in both PLRG1-deficient MEFs and zebrafish in vivo, showing that apoptosis resulting from PLRG1 deficiency is p53 dependent. Moreover, the deletion of PLRG1 results in the relocation of its interaction partner CDC5L from the nucleus to the cytoplasm without general alterations in pre-mRNA splicing. Taken together, the results of this study identify PLRG1 as a critical nuclear regulator of p53-dependent cell cycle progression and apoptosis during both embryonic development and adult tissue homeostasis.Mammalian pleiotropic regulator (PLRG1) belongs to a highly conserved family of seven WD40 domain-containing proteins in eukaryotes (2, 4). The founding members of this protein family, PRL1 and PRL2, were identified first by T-DNA tagging in Arabidopsis thaliana, where PRL1 deficiency augments the sensitivity to growth hormones, stimulates the accumulation of sugars and starch in leaves, and inhibits root elongation (37). More recently, it has been demonstrated that the A. thaliana CDC5/PRL1 complex is essential for plant innate immunity (39).Human PLRG1 initially was identified as a subunit of spliceosomal complexes purified from HeLa nuclear extracts by coimmunoprecipitation with CDC5L (2). Indeed, interaction between PLRG1 and CDC5L appears essential for pre-mRNA processing, as peptides inhibiting CDC5L-PLRG1 complex formation efficiently block pre-mRNA splicing (3).The CDC5L-PLRG1 complex also was found to interact with the WRN protein, which is deficient in Werner syndrome, a rare autosomal recessive human disorder characterized by genomic instability and the premature onset of a number of age-related diseases, including cancer (1,18,28,34,53). The WRN protein has exonuclease and RecQ helicase activity, interacts with multiple proteins of the DNA replication complex, and is crucial for DNA synthesis and DNA repair (12,30,31,41,43,53). Thus, PLRG1 offers an interesting link between the control of pre-mRNA splicing and DNA metabolism.To elucidate the in vivo role of PLRG1 in vertebrates, we have generated conventional and conditional knockout (KO) mice for PLRG1, as well as antisense-mediated plrg1 knockdown zebrafish embryos and cell lines with an inducible inactivation of PLRG1...

SON is a spliceosome-associated factor required for mitotic progression

“…Similarly, inactivation of spliceosome factors SC-35 and PLRG1 coincided with elevated DNA damage and genome instability. 6,7 Together, these observations lend credence to the idea that splicing factors may, directly or indirectly, modulate cell the eukaryotic RNA splicing machinery is dedicated to the daunting task of excising intronic sequences on the many nascent RNA transcripts in a cell, and in doing so facilitates proper translation of its transcriptome. Notably, emerging evidence suggests that RNA splicing may also play direct roles in maintaining genome stability.…”

BCAS2 is essential for Drosophila viability and functions in pre-mRNA splicing

“…Previous reports have illustrated that depletion of spliceosomal factors, such as ASF and dMFAP1, cause G2/M arrest (Bernstein and Coughlin 1998;Li et al 2005;Andersen and Tapon 2008). Similarly, other members of the hPrp19 complex, hPrp19 and PLRG1, which are both required for RNA splicing, caused lethality at an early stage of embryo development when knocked out in mice (Fortschegger et al 2007;Kleinridders et al 2009). These reports support our data that the impairment of RNA splicing when BCAS2 is knocked down in p53-null cells causes lethality or malformations in dBCAS2-depleted flies.…”

Graded requirement for the spliceosome in cell cycle progression

“…17 Different snRNPs and accessory proteins are recruited or excluded at different steps of the splicing process, resulting in a very dynamic composition of the spliceosome machine and the generation of extensive rearrangements during different stages of splicing. 17,18 A connection between the spliceosome and cell cycle progression has been found in many organisms including budding yeast, [19][20][21][22][23][24] fission yeast, [25][26][27] Drosophila, 9,28 chicken, 29 mouse, 30 and human cells. 6,11,12,29,31,32 In human cells, depletion of different spliceosome components with siRNAs results in multiple cell cycle defects, with most siRNAs analyzed eliciting mitotic defects 6,11,12,31 although accumulation of cells in S phase 32 has also been observed.…”

“…Similarly, mouse cells with the spliceosome component PLRG1 deleted show an increase in DNA damage with no evidence of major splicing defects. 30 However, unlike the G2 arrest observed after ASF/SF2 depletion, 29 PLRG1 deficiency causes an earlier cell cycle arrest, as cells cannot progress through S phase. 30 Hence, a connection between spliceosome depletion and cell cycle defects has been observed after depletion of many different spliceosome subunits.…”

“…30 However, unlike the G2 arrest observed after ASF/SF2 depletion, 29 PLRG1 deficiency causes an earlier cell cycle arrest, as cells cannot progress through S phase. 30 Hence, a connection between spliceosome depletion and cell cycle defects has been observed after depletion of many different spliceosome subunits. However, the specific cell cycle phenotype observed varies depending on the spliceosome subunit targeted and the type of cells used.…”