Effects of the Bowen-Conradi syndrome mutation in EMG1 on its nuclear import, stability and nucleolar recruitment

Abstract

Bowen-Conradi syndrome (BCS) is a severe genetic disorder that is characterised by various developmental abnormalities, bone marrow failure and early infant death. This disease is caused by a single mutation leading to the aspartate 86 to glycine (D86G) exchange in the essential nucleolar RNA methyltransferase EMG1. EMG1 is required for the synthesis of the small ribosomal subunit and is involved in modification of the 18S ribosomal RNA. Here, we identify the pre-ribosomal factors NOP14, NOC4L and UTP14A as members of a nucleolar subcomplex that contains EMG1 and is required for its recruitment to nucleoli. The BCS mutation in EMG1 leads to reduced nucleolar localisation, accumulation of EMG1D86G in nuclear foci and its proteasome-dependent degradation. We further show that EMG1 can be imported into the nucleus by the importins (Imp) Impα/β or Impβ/7. Interestingly, in addition to its role in nuclear import, binding of the Impβ/7 heterodimer can prevent unspecific aggregation of both EMG1 and EMG1D86G on RNAs in vitro, indicating that the importins act as chaperones by binding to basic regions of the RNA methyltransferase. Our findings further indicate that in BCS, nuclear disassembly of the import complex and release of EMG1D86G lead to its nuclear aggregation and degradation, resulting in the reduced nucleolar recruitment of the RNA methyltransferase and defects in the biogenesis of the small ribosomal subunit.

The m6A reader protein YTHDC2 interacts with the small ribosomal subunit and the 5′–3′ exoribonuclease XRN1

“…Immunofluorescence was essentially performed as previously described (Warda et al 2016). In brief, HeLa cells or HEK293 cells expressing YTHDC2-Flag were grown on coverslips and fixed using 4% paraformaldehyde in PBS for 10 min at RT.…”

The mitochondrial epitranscriptome: the roles of RNA modifications in mitochondrial translation and human disease

“…Notably, its catalytic function on 12S rRNA is independent of MTERF4 and lack of NSUN4 (or m 5 C841) does not affect the installation the 12S- and modifications, implying that it is not essential for assembly of the SSU. Several other methyltransferases involved in rRNA modification have also been demonstrated to have dual functions [ 25 , 40 , 54 , 55 ]. However, these enzymes are typically required for different aspects of the biogenesis of a single ribosomal subunit, but in the case of NSUN4, it is possible that its functions in both SSU modification and LSU assembly may represent a mechanism for co-ordinating maturation of both ribosomal subunits [ 44 ].…”

The human RNA helicase DHX37 is required for release of the U3 snoRNP from pre-ribosomal particles

“…Immunofluorescence was essentially performed as previously described in [19]. HeLa cells were grown on coverslips before fixing using 4% paraformaldehyde in PBS for 10 min at room temperature.…”

“…A subset of RPs and RBFs are recruited to the nascent pre-rRNA transcript giving rise to an early pre-ribosomal complex termed the small subunit (SSU) processome. The SSU processome is composed of numerous sub-complexes, including the UTP-A, UTP-B and UTP-C complexes, as well as various enzymes that mediate important steps in SSU biogenesis, such as the endonuclease UTP24, the PIN domain protein UTP23, the rRNA methyltransferase EMG1 and the GTPase BMS1 [6,[17][18][19]. A core component of the SSU processome is the U3 snoRNP, which in contrast to most other snoRNPs, does not guide rRNA modification, but instead bridges key interactions within early pre-ribosomes [20].…”