An allelic series for studying the mouse Thoc1 gene

Abstract

Thoc1 encodes an essential component of the mammalian TREX protein complex. TREX is an evolutionary conserved complex that couples elongating RNA polymerase II with RNA processing factors. Depletion of Thoc1 protein (pThoc1) compromises transcriptional elongation and nuclear export of some RNAs. Loss of Thoc1 causes periimplantation embryonic lethality in the mouse. Early embryonic lethality precludes analysis of the physiological requirements for Thoc1 in the developing embryo or adult. To circumvent this limitation, we have generated mice containing hypomorphic or conditional alleles of Thoc1. Mice homozygous for the conditional allele appear normal. Mice containing Cre recombined conditional alleles phenocopy the previously characterized Thoc1 null allele. Mice homozygous for the hypomorphic allele are viable and born at a frequency that is not significantly different from the expected Mendelian ratio. However, these mice express less pThoc1 than wild type mice and exhibit a dwarf phenotype. The dwarf phenotype can be detected in midgestation embryos, suggesting that Thoc1 is also required later in embryonic and postnatal development.Keywords gene expression; development; RNA processing; mRNP Co-transcriptional loading of RNA processing and export factors onto nascent RNA supports efficient and regulated gene expression (Moore, 2005;Reed, 2003). Assembly of the TREX complex is one molecular mechanism utilized by cells to physically couple transcription with factors involved in RNA processing and export. TREX has been best characterized in S. cerevisiae where it is composed of the THO sub-complex containing four proteins (Hpr1p, Tho2p, Mft1p, and Thp2p) that are essential for normal transcriptional elongation and genome stability (Chavez et al., 2000). The THO sub-complex recruits two additional proteins, Sub2p and Yra1p, that are involved in nuclear RNA export and splicing (Luo et al., 2001;Strasser et al., 2002). Hpr1p genetically and physically interacts with RNA PolII (Chang et al., 1999;Chavez et al., 2000;Fan et al., 1996;Strasser et al., 2002). Hpr1p is an essential component of TREX since its loss impairs both transcriptional elongation and nuclear RNA export (Chavez and Aguilera, 1997;Chavez et al., 2001;Fan et al., 1996;Huertas and Aguilera, 2003;Rondon et al., 2003;Strasser et al., 2002;Zenklusen et al., 2002). Despite the defects in gene expression associated with HPR1 loss, it is not essential for yeast viability (Aguilera and Klein, 1990).Metazoan structural homologues are apparent for the yeast TREX proteins Tho2p (Thoc2), Sub2p (UAP56), and Yra1p (Aly) (Luo et al., 2001;Strasser and Hurt, 2000;Stutz et al., 2000;West et al., 2000). 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 blastoc...

The THO Ribonucleoprotein Complex Is Required for Stem Cell Homeostasis in the Adult Mouse Small Intestine

“…Inducible Cre recombinase-mediated Thoc5 gene deletion has been reported to affect hematopoiesis, but effects on other tissues are unknown because of the limited distribution of tissues suffering Thoc5 deficiency (20). Mice homozygous for a hypomorphic Thoc1 allele, which expresses reduced Thoc1 protein in many tissues, are viable but sterile (19,25). This supports the hypothesis that different tissues have different requirements for Thoc1 during development.…”

Cancer Cells and Normal Cells Differ in Their Requirements for Thoc1

“…We have recently generated a conditionally excisable allele of the murine Thoc1 gene that facilitates stable depletion of pThoc1 (26). The modified Thoc1 allele (Thoc1F) contains lox P sites within introns 5 and 7.…”

“…Murine embryonic fibroblast (MEF) cultures were isolated from F/À and F/+ littermate embryos at 13.5 days of gestation by standard procedures. The construction and genotyping of the floxed Thoc1 allele has previously been described (26). Cells were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamine, and 0.5% penicillin and streptomycin and grown at 37jC in a humidified atmosphere of 5% CO 2 .…”

Characterization of EMU, the Arabidopsis homolog of the yeast THO complex member HPR1

“…Recently, a role for mouse Thoc1/ Hpr1/p84 during embryogenesis was revealed using mice containing the mutant alleles (Wang et al 2006(Wang et al , 2007, and researchers have now started to analyze THO components in a physiological context in multicellular organisms. In the present study we showed that mutations in EMU and AtTHO2 compromise Arabidopsis development.…”