Cell preparations were fixed in PFA without pre-extraction (scale bar 5 m)

Cell preparations were fixed in PFA without pre-extraction (scale bar 5 m). suggests that defects in RNP biogenesis pathways contribute to the pathology of HHS. INTRODUCTION Regulator of Telomere ELongation helicase 1 (RTEL1) was first identified in as being responsible for the maintenance of long telomeres in embryonic stem (ES) cells (1). Mouse Rtel1 is involved in telomere replication, but also in genome wide replication, presumably by facilitating the progression of the replication fork (2,3). Mouse Rtel1 is also required for genome stability and repair (4) and the human protein has been suggested to restrict recombination through IDE1 a helicase activity that dismantles recombination intermediate substrates (5,6). That human RTEL1 is involved in telomere metabolism has been recently confirmed by the identification of mutations in patients with Hoyeraal-Hreidarsson syndrome (HHS), a severe form of dyskeratosis congenita characterized by short telomeres, developmental defects, bone marrow failure and immunodeficiency (7C11). However, the precise role of human RTEL1 remains largely speculative. Based on results obtained from an initially unbiased analysis of potential human RTEL1 interactors, we set out to explore the role of this protein in non-coding RNA metabolism, both experimentally and in the context of HHS. We discovered that RTEL1 is required for the normal export of Mouse monoclonal to TRX pre-U2 small nuclear (sn) RNA to the cytoplasm and for its trafficking through that compartment before being re-imported to the nucleus. U2 is a key component of the major spliceosome (12). It is transcribed by POLII as a precursor containing a 3 extension (13). As soon as this snRNA is produced, its methylated cap is bound by the cap-binding complex, CBC (14), which is in turn bound by an activated (phosphorylated) form of phosphorylated adaptor for RNA export (PHAX) (15). PHAX is a mediator of snRNA export through its interaction with XPO1 (CRM1) (15). In the cytoplasm, the ribonucleotide protein (RNP) export complex dissociates and pre-U2 is transferred to the survival motor neuron (SMN) complex through its interaction with GEMIN5, another cap-binding protein (16). Once the IDE1 pre-U2 snRNA is loaded onto the SMN complex, the assembly machine for spliceosomal IDE1 RNPs (17), it undergoes maturation through both removal of the last 11C12 nucleotides and trimethylation of its cap, before being re-imported to the nucleus (17,18). Our results show that RTEL1 is required for the export of the pre-U2 RNP complex to the cytoplasm. Furthermore, mutations in the RING domain of the protein (13), carried by some HHS patients, are responsible for defects in the cytoplasmic trafficking of the pre-U2 RNP. Finally, we show that expression of mutated forms of RTEL1 impact the efficiency of splicing reactions, in agreement with a defect in U2 RNA biogenesis, and which strongly suggests that these defects may be responsible for at least some of the clinical manifestations and contribute to the severity of the disease phenotype in HHS patients. MATERIALS AND METHODS Cloning of RTEL1 cDNA and preparations of DNA constructs For mammalian expression FL RTEL1 (nt 1C3903) was amplified from HeLa cells and was cloned as an EcoRI/HindIII fragment into pCMV-Tag2B (Stratagene), which IDE1 contains an IRES-EGFP inserted as an XhoI/PacI fragment. The mutant RTEL1 W (eliminating residues 34C48), and the Cter mutant (nt 1C3492) were created by polymerase chain reaction (PCR) and confirmed by sequencing (The sequences of primers used are available upon request). The RING domain was mutated by site directed mutagenesis using the QuickChange kit (Stratagene) and the primers top: 5-TGACTTCCAGCGCGGCCAAGCCGGCTGGCAACGGCA.-3, bottom: 5-TGCCGTTGCCAGCCGGCTTGGCCGCGCTGGAAGTCA-3; the amino acid modifications for the mutated RING (mR) are C1265A and C1268A. Mutating the NLS domain was done as follows: to generate the mutations R875A, K876A, K877A PCR was performed with the forward primer 5-GCGGCCGCGATCCGGCTGGTCAGCCACCC-3 and downstream primer, and with a reverse IDE1 primer 5-GCGGCCGCCCCTCCTCGCGGTTCTTCTGC-3 and an upstream primer; in the process a NotI.