Thoc1/Hpr1/p84 Is Essential for Early Embryonic Development in the Mouse

Abstract

The yeast TREX complex physically couples elongating RNA polymerase II with RNA processing and nuclear RNA export factors to facilitate regulated gene expression. Hpr1p is an essential component of TREX, and loss of Hpr1p compromises transcriptional elongation, RNA export, and genome stability. Despite these defects, HPR1 is not essential for viability in yeast. A functional orthologue of Hpr1p has been identified in metazoan species and is variously known as Thoc1, Hpr1, or p84. However, the physiological functions of this protein have not been determined. Here, we describe the generation and phenotypic characterization of mice containing a null allele of the Thoc1 gene. Heterozygous null Thoc1 mice are born at the expected Mendelian frequency with no phenotype distinguishable from the wild type. In contrast, homozygous null mice are not recovered, indicating that Thoc1 is required for embryonic development. Embryonic development is arrested around the time of implantation, as blastocysts exhibit hatching and blastocyst outgrowth defects upon in vitro culture. Cells of the inner cell mass are particularly dependent on Thoc1, as these cells rapidly lose viability coincident with Thoc1 protein loss. While Hpr1p is not essential for the viability of unicellular yeasts, the orthologous Thoc1 protein is required for viability of the early mouse embryo.Cotranscriptional loading of RNA-processing and export factors onto nascent RNA supports efficient and regulated gene expression (15,17). Assembly of the TREX complex is one molecular mechanism utilized in yeast to physically couple factors involved in transcriptional elongation with those important for RNA splicing and export. TREX is composed of the THO subcomplex containing four proteins (Hpr1p, Tho2p, Mft1p, and Thp2p) that are essential for normal transcriptional elongation of some yeast genes (3). THO associates with two proteins implicated in nuclear RNA export (Sub2p and Yra1) to form the larger TREX complex (23). Sub2p is the yeast orthologue of human UAP56, which has also been implicated in RNA splicing (12). Hpr1p genetically and physically interacts with RNA polymerase II (1,3,6,23) and is essential for recruitment of Sub2p to genes regulated by TREX (27). Loss of Hpr1p impairs both transcriptional elongation and nuclear RNA export (2,4,6,9,19,23). Hpr1p, therefore, is an essential component of the THO and TREX complexes. Hpr1p has also been associated with the Paf1 complex, which influences transcriptional elongation through alterations in histone methylation (1,10,16,26). Despite the defects in gene expression associated with loss of Hpr1p, it is not essential for viability in yeast. However, hpr1⌬ yeasts are temperature sensitive for growth (7).Metazoan species with long, intron-containing genes are likely even more dependent on the physical coupling of transcriptional elongation and RNA processing for efficient gene expression. The presence of metazoan structural homologues for the yeast TREX proteins Tho2p (Thoc2), Sub2p (UAP56), and Yra1p (Aly) sugges...

An allelic series for studying the mouse Thoc1 gene

“…However, they were unable to form blastocyst outgrowths upon in vitro culture (Fig. 4A), phenocopying previously characterized homozygous Thoc1 null blastocysts (Wang et al, 2006). Treatment of MEFs hemizygous for the F allele with a recombinant adenovirus designed to express Cre significantly depleted pThoc1 levels relative to control MEFs containing a wild type allele (Fig.…”

“…Although lacking statistically significant similarity at the primary amino acid level, functional orthologues of yeast HPR1 have been identified in both human and insect cells (alternatively known as Thoc1, Hpr1, or p84) (Li et al, 2005; Thoc1 is expressed widely during mouse embryonic development and mice lacking Thoc1 fail to develop past the late blastocyst stage (Wang et al, 2006). Early embryonic lethality is associated with loss of cell viability, particularly among the pluripotent stem cells that make up the inner cell mass.…”

“…Viable neonates hemizygous for the Thoc1 H allele (H/−) were not recovered (Table 1). However, hemizygous embryos were detected at E13.5, thus reaching a stage of embryonic development significantly more advanced than Thoc1 null embryos (Wang et al, 2006 Mice homozygous, heterozygous, or hemizygous for the F allele were born at the expected Mendelian ratio (Table 1). Mice with these genotypes exhibited no phenotype distinguishable from wild type mice to at least nine months of age.…”

“…PCR assays have been designed for routine genotyping of the H, F, and the previously described Thoc1 null alleles ( Fig. 2A-B) (Wang et al, 2006). Cre expression correctly excised exons 6 and 7 from the F allele to generate a new, presumed null allele (N) as indicated by the appearance of a PCR amplified fragment of expected size (Fig.…”

“…Consistent with Cre mediated generation of a premature stop codon, pThoc1 levels detected by a carboxy terminal specific antibody f are significantly reduced in F/− MEFs exposed to Cre recombinase. The Cre recombined N allele phenocopies the previously characterized null allele since N/N embryos fail to develop beyond the blastocyst stage (Wang et al, 2006). Due to the perimplantation embryonic lethality of Thoc1 null mice, these two new alleles will be useful in studying Thoc1 function during later stages of development and in adult mice.…”