Janine Altmueller

NSUN4 Is a Dual Function Mitochondrial Protein Required for Both Methylation of 12S rRNA and Coordination of Mitoribosomal Assembly

Biogenesis of mammalian mitochondrial ribosomes requires a concerted maturation of both the small (SSU) and large subunit (LSU). We demonstrate here that the m5C methyltransferase NSUN4, which forms a complex with MTERF4, is essential in mitochondrial ribosomal biogenesis as mitochondrial translation is abolished in conditional Nsun4 mouse knockouts. Deep sequencing of bisulfite-treated RNA shows that NSUN4 methylates cytosine 911 in 12S rRNA (m5C911) of the SSU. Surprisingly, NSUN4 does not need MTERF4 to generate this modification. Instead, the NSUN4/MTERF4 complex is required to assemble the SSU and LSU to form a monosome. NSUN4 is thus a dual function protein, which on the one hand is needed for 12S rRNA methylation and, on the other hand interacts with MTERF4 to facilitate monosome assembly. The presented data suggest that NSUN4 has a key role in controlling a final step in ribosome biogenesis to ensure that only the mature SSU and LSU are assembled.

CLUH regulates mitochondrial biogenesis by binding mRNAs of nuclear-encoded mitochondrial proteins

CLUH is a cytosolic mRNA-binding protein that specifically binds a subset of mRNAs encoding mitochondrial proteins and may regulate their localized translation.

Transcriptome Analysis of Murine Olfactory Sensory Neurons during Development Using Single Cell RNA-Seq

Mammalian odor reception is achieved by highly specialized olfactory sensory neurons (OSNs) located in the nasal cavity. Despite their importance for the daily survival of most mammals, the gene expression and regulatory profiles of these single neurons are poorly understood. Here, we report the isolation of individual GFP-labeled OSNs from Olfr73-GFP mice at different developmental stages followed by Next Generation Sequencing, thereby analyzing the detailed transcriptome for the first time. We characterized the repertoire of olfactory receptors (ORs) and found that in addition to the highly and predominant detectable Olfr73, 20 additional ORs were stably detectable at lower transcript levels in adult mice. Additionally, OSNs collected from mice of earlier developmental stages did not show any stable OR patterns. However, more than one predominant OR per OSN was detectable.

Label-Free Protein-RNA Interactome Analysis Identifies Khsrp Signaling Downstream of the p38/Mk2 Kinase Complex as a Critical Modulator of Cell Cycle Progression

Growing evidence suggests a key role for RNA binding proteins (RBPs) in genome stability programs. Additionally, recent developments in RNA sequencing technologies, as well as mass-spectrometry techniques, have greatly expanded our knowledge on protein-RNA interactions. We here use full transcriptome sequencing and label-free LC/MS/MS to identify global changes in protein-RNA interactions in response to etoposide-induced genotoxic stress. We show that RBPs have distinct binding patterns in response to genotoxic stress and that inactivation of the RBP regulator module, p38/MK2, can affect the entire spectrum of protein-RNA interactions that take place in response to stress. In addition to validating the role of known RBPs like Srsf1, Srsf2, Elavl1 in the genotoxic stress response, we add a new collection of RBPs to the DNA damage response. We identify Khsrp as a highly regulated RBP in response to genotoxic stress and further validate its role as a driver of the G1/S transition through the suppression of Cdkn1aP21 transcripts. Finally, we identify KHSRP as an indicator of overall survival, as well as disease free survival in glioblastoma multiforme.

CWC22-dependent pre-mRNA splicing and eIF4A3 binding enables global deposition of exon junction complexes

In metazoan cells, spliced mRNAs are marked by the exon junction complex (EJC), a multi-protein complex that serves as a key regulator of post-transcriptional mRNA metabolism. Deposition of EJCs on mRNA is intimately linked to the splicing process. The spliceosomal protein CWC22 directly binds the core EJC-protein eIF4A3, guides it to the spliceosome and initiates EJC assembly. In addition, CWC22 is involved in the splicing process itself, but the molecular details of its dual function remain elusive. Here we analyze the mechanisms, by which CWC22 co-regulates pre-mRNA splicing and EJC assembly. We show that the core of CWC22 is sufficient to mediate both pre-mRNA splicing and EJC assembly. Nonetheless, both processes can be functionally uncoupled with an eIF4A3-binding deficient mutant of CWC22, which impedes EJC assembly. A C-terminal domain of CWC22 strongly enhances its spliceosomal interaction and likely regulates its function. High-throughput RNA-sequencing identifies global defects of pre-mRNA splicing and downregulation of diverse gene expression pathways in CWC22-depleted cells. We propose a model, in which CWC22 represents an integral component of the spliceosome and orchestrates pre-mRNA splicing and eIF4A3 binding to achieve global assembly of exon junction complexes.

Identification of Novel and Recurrent Disease-Causing Mutations in Retinal Dystrophies Using Whole Exome Sequencing (WES): Benefits and Limitations

Inherited retinal dystrophies (IRDs) are Mendelian diseases with tremendous genetic and phenotypic heterogeneity. Identification of the underlying genetic basis of these dystrophies is therefore challenging. In this study we employed whole exome sequencing (WES) in 11 families with IRDs and identified disease-causing variants in 8 of them. Sequence analysis of about 250 IRD-associated genes revealed 3 previously reported disease-associated variants in RHO, BEST1 and RP1. We further identified 5 novel pathogenic variants in RPGRIP1 (p.Ser964Profs*37), PRPF8 (p.Tyr2334Leufs*51), CDHR1 (p.Pro133Arg and c.439-17G>A) and PRPF31 (p.Glu183_Met193dup). In addition to confirming the power of WES in genetic diagnosis of IRDs, we document challenges in data analysis and show cases where the underlying genetic causes of IRDs were missed by WES and required additional techniques. For example, the mutation c.439-17G>A in CDHR1 would be rated unlikely applying the standard WES analysis. Only transcript analysis in patient fibroblasts confirmed the pathogenic nature of this variant that affected splicing of CDHR1 by activating a cryptic splice-acceptor site. In another example, a 33-base pair duplication in PRPF31 missed by WES could be identified only via targeted analysis by Sanger sequencing. We discuss the advantages and challenges of using WES to identify mutations in heterogeneous diseases like IRDs.

Deep ion sequencing of amplicon adapter ligated libraries: a novel tool in molecular diagnostics of formalin fixed and paraffin embedded tissues

Due to the advanced progress in personalised therapy concepts for non-small cell lung cancer (NSCLC), we applied the ion semiconductor sequencing (ISS) approach to molecular diagnosis of NSCLC, analysing a set of therapy relevant gene loci. DNA from macrodissected tumour samples of formalin fixed biopsies was used for PCR amplification of EGFR exons 18, 19, 21 and KRAS exon 1. A total of 128 PCR products were analysed by conventional termination sequencing as well as by ISS. Sensitivity of ISS was additionally determined using 100–10 000 copies of reference mutants. All somatic mutations detected by direct Sanger sequencing were also identified by ISS. No additional mutants were detected. Running samples with limited copies of mutated alleles revealed high sensitivity, detecting less than 10% (2500 copies) mutants in a human wild type background. In conclusion, multiplexed mutation analyses by ISS is an efficient technology that can easily be linked to existing PCR approaches in molecular pathology.

Single molecule real time sequencing in ADTKD- MUC1 allows complete assembly of the VNTR and exact positioning of causative mutations

Recently, the Mucin-1 (MUC1) gene has been identified as a causal gene of autosomal dominant tubulointerstitial kidney disease (ADTKD). Most causative mutations are buried within a GC-rich 60 basepair variable number of tandem repeat (VNTR), which escapes identification by massive parallel sequencing methods due to the complexity of the VNTR. We established long read single molecule real time sequencing (SMRT) targeted to the MUC1-VNTR as an alternative strategy to the snapshot assay. Our approach allows complete VNTR assembly, thereby enabling the detection of all variants residing within the VNTR and simultaneous determination of VNTR length. We present high resolution data on the VNTR architecture for a cohort of snapshot positive (n = 9) and negative (n = 7) ADTKD families. By SMRT sequencing we could confirm the diagnosis in all previously tested cases, reconstruct both VNTR alleles and determine the exact position of the causative variant in eight of nine families. This study demonstrates that precise positioning of the causative mutation(s) and identification of other coding and noncoding sequence variants in ADTKD-MUC1 is feasible. SMRT sequencing could provide a powerful tool to uncover potential factors encoded within the VNTR that associate with intra- and interfamilial phenotype variability of MUC1 related kidney disease.

Topological Demarcation By HMGB2 Is Disrupted Early Upon Senescence Entry Across Cell Types And Induces CTCF Clustering

Ageing-relevant processes, like cellular senescence, are characterized by complex, often stochastic, events giving rise to heterogeneous cell populations. We hypothesized that entry into senescence of different primary human cells can be triggered by one early molecular event affecting the spatial organization of chromosomes. To test this, we combined whole-genome chromosome conformation capture, population and single-cell transcriptomics, super-resolution imaging, and functional analyses applied on proliferating and replicatively-senescent populations from three distinct human cell types. We found a number of genes involved in DNA conformation maintenance being suppressed upon senescence across cell types. Of these, the abundant high mobility group (HMG) B1 and B2 nuclear factors are quantitatively removed from cell nuclei before typical senescence markers appear, and mark a subset of topologically-associating domain (TAD) boundaries. Their loss coincides with obvious reorganization of chromatin interactions via the dramatic spatial clustering of CTCF foci. HMGB2 knock-down recapitulates this senescence-induced CTCF clustering, while also affecting insulation at TAD boundaries. We accordingly propose that HMGB-mediated deregulation of chromosome conformation constitutes a primer for the ensuing senescent program across cell types.

Targeted sequencing with expanded gene profile enables high diagnostic yield in non-5q-spinal muscular atrophies

Spinal muscular atrophies (SMAs) are a heterogeneous group of disorders characterized by muscular atrophy, weakness, and hypotonia due to suspected lower motor neuron degeneration (LMND). In a large cohort of 3,465 individuals suspected with SMA submitted for SMN1 testing to our routine diagnostic laboratory, 48.8% carried a homozygous SMN1 deletion, 2.8% a subtle mutation, and an SMN1 deletion, whereas 48.4% remained undiagnosed. Recently, several other genes implicated in SMA/LMND have been reported. Despite several efforts to establish a diagnostic algorithm for non‐5q‐SMA (SMA without deletion or point mutations in SMN1 [5q13.2]), data from large‐scale studies are not available. We tested the clinical utility of targeted sequencing in non‐5q‐SMA by developing two different gene panels. We first analyzed 30 individuals with a small panel including 62 genes associated with LMND using IonTorrent‐AmpliSeq target enrichment. Then, additional 65 individuals were tested with a broader panel encompassing up to 479 genes implicated in neuromuscular diseases (NMDs) with Agilent‐SureSelect target enrichment. The NMD panel provided a higher diagnostic yield (33%) than the restricted LMND panel (13%). Nondiagnosed cases were further subjected to exome or genome sequencing. Our experience supports the use of gene panels covering a broad disease spectrum for diseases that are highly heterogeneous and clinically difficult to differentiate.