Tatsuaki Kurosaki

Nonsense-mediated mRNA decay in humans at a glance.

ABSTRACT Nonsense-mediated mRNA decay (NMD) is an mRNA quality-control mechanism that typifies all eukaryotes examined to date. NMD surveys newly synthesized mRNAs and degrades those that harbor a premature termination codon (PTC), thereby preventing the production of truncated proteins that could result in disease in humans. This is evident from dominantly inherited diseases that are due to PTC-containing mRNAs that escape NMD. Although many cellular NMD targets derive from mistakes made during, for example, pre-mRNA splicing and, possibly, transcription initiation, NMD also targets ∼10% of normal physiological mRNAs so as to promote an appropriate cellular response to changing environmental milieus, including those that induce apoptosis, maturation or differentiation. Over the past ∼35 years, a central goal in the NMD field has been to understand how cells discriminate mRNAs that are targeted by NMD from those that are not. In this Cell Science at a Glance and the accompanying poster, we review progress made towards this goal, focusing on human studies and the role of the key NMD factor up-frameshift protein 1 (UPF1). Summary: In humans, nonsense-mediated mRNA decay prevents the synthesis of potentially toxic proteins from mutated mRNAs, and also regulates 10% of physiological mRNAs that promote cellular adaptation to changing environments.

Rules that govern UPF1 binding to mRNA 3′ UTRs

Nonsense-mediated mRNA decay (NMD), which degrades transcripts harboring a premature termination codon (PTC), depends on the helicase up-frameshift 1 (UPF1). However, mRNAs that are not NMD targets also bind UPF1. What governs the timing, position, and function of UPF1 binding to mRNAs remains unclear. We provide evidence that (i) multiple UPF1 molecules accumulate on the 3′-untranslated region (3′ UTR) of PTC-containing mRNAs and to an extent that is greater per unit 3′ UTR length if the mRNA is an NMD target; (ii) UPF1 binding begins ≥35 nt downstream of the PTC; (iii) enhanced UPF1 binding to the 3′ UTR of PTC-containing mRNA relative to its PTC-free counterpart depends on translation; and (iv) the presence of a 3′ UTR exon–junction complex (EJC) further enhances UPF1 binding and/or affinity. Our data suggest that NMD involves UPF1 binding along a 3′ UTR whether the 3′ UTR contains an EJC. This binding explains how mRNAs without a 3′ UTR EJC but with an abnormally long 3′ UTR can be NMD targets, albeit not as efficiently as their counterparts that contain a 3′ UTR EJC.

The neuronal POU transcription factor Brn-2 interacts with Jab1, a gene involved in the onset of neurodegenerative diseases.

Brain-2 (Brn-2), a Class III POU transcription factor, plays an important role in the development of the neocortex and the establishment of neural cell lineage. Here we performed a yeast two-hybrid screening to identify the Brn-2 binding proteins. We obtained Jun-activation-domain-binding protein 1 (Jab1) as a new Brn-2 binding protein. Direct interaction between Brn-2 and Jab1 was confirmed by using a surface plasmon resonance biosensor. Considering the involvement of Jab1 in the onset of the neurodegenerative disorders such as Parkinsons disease and Alzheimers disease, the interaction between Brn-2 and Jab1 may provide some insights into the understanding of neuronal development and neurodegenerative diseases.

The Unstable CCTG Repeat Responsible for Myotonic Dystrophy Type 2 Originates from an AluSx Element Insertion into an Early Primate Genome

Myotonic dystrophy type 2 (DM2) is a subtype of the myotonic dystrophies, caused by expansion of a tetranucleotide CCTG repeat in intron 1 of the zinc finger protein 9 (ZNF9) gene. The expansions are extremely unstable and variable, ranging from 75–11,000 CCTG repeats. This unprecedented repeat size and somatic heterogeneity make molecular diagnosis of DM2 difficult, and yield variable clinical phenotypes. To better understand the mutational origin and instability of the ZNF9 CCTG repeat, we analyzed the repeat configuration and flanking regions in 26 primate species. The 3′-end of an AluSx element, flanked by target site duplications (5′-ACTRCCAR-3′or 5′-ACTRCCARTTA-3′), followed the CCTG repeat, suggesting that the repeat was originally derived from the Alu element insertion. In addition, our results revealed lineage-specific repetitive motifs: pyrimidine (CT)-rich repeat motifs in New World monkeys, dinucleotide (TG) repeat motifs in Old World monkeys and gibbons, and dinucleotide (TG) and tetranucleotide (TCTG and/or CCTG) repeat motifs in great apes and humans. Moreover, these di- and tetra-nucleotide repeat motifs arose from the poly (A) tail of the AluSx element, and evolved into unstable CCTG repeats during primate evolution. Alu elements are known to be the source of microsatellite repeats responsible for two other repeat expansion disorders: Friedreich ataxia and spinocerebellar ataxia type 10. Taken together, these findings raise questions as to the mechanism(s) by which Alu-mediated repeats developed into the large, extremely unstable expansions common to these three disorders.

LDB3 splicing abnormalities are specific to skeletal muscles of patients with myotonic dystrophy type 1 and alter its PKC binding affinity.

Myotonic dystrophy type 1 (DM1) is caused by transcription of CUG repeat RNA, which causes sequestration of muscleblind-like 1 (MBNL1) and upregulation of CUG triplet repeat RNA-binding protein (CUG-BP1). In DM1, dysregulation of these proteins contributes to many aberrant splicing events, causing various symptoms of the disorder. Here, we demonstrate the occurrence of aberrant splicing of LIM domain binding 3 (LDB3) exon 11 in DM1 skeletal muscle. Exon array surveys, RT-PCR, and western blotting studies demonstrated that exon 11 inclusion was DM1 specific and could be reproduced by transfection of a minigene containing the CTG repeat expansion. Moreover, we found that the LDB3 exon 11-positive isoform had reduced affinity for PKC compared to the exon 11-negative isoform. Since PKC exhibits hyperactivation in DM1 and stabilizes CUG-BP1 by phosphorylation, aberrant splicing of LDB3 may contribute to CUG-BP1 upregulation through changes in its affinity for PKC.

Exome sequencing as a diagnostic tool to identify a causal mutation in genetically highly heterogeneous limb-girdle muscular dystrophy

Exome sequencing as a diagnostic tool to identify a causal mutation in genetically highly heterogeneous limb-girdle muscular dystrophy

Comparative Genetics of the Poly-Q Tract of Ataxin-1 and Its Binding Protein PQBP-1

Human PQBP-1 is known to interact with triplet repeat disease gene products such as ataxin and huntingtin through their poly-glutamine (poly-Q) tracts. The poly-Q tracts show extensive variation in both the number and the configuration of repeats among species. A surface plasmon resonance assay showed clear interaction between human PQBP-1 and Q11, representative of the poly-Q tract of the ataxin-1 of Old World monkeys. No response was observed using Q2PQ2P4Q2, representative of the poly-Q tract of the ataxin-1 of New World monkeys. This implies that the interaction of human PQBP-1 with ataxin-1 is limited to humans and closely related species. Comparison of the human and mouse PQBP-1 sequences showed an elevated amino acid substitution rate in the polar amino acid-rich domain of PQBP-1 that is responsible for binding to poly-Q tracts. This could have been advantageous to the new biological function of human PQBP-1 through poly-Q tracts.

Identifying Cellular Nonsense-Mediated mRNA Decay (NMD) Targets: Immunoprecipitation of Phosphorylated UPF1 Followed by RNA Sequencing (p-UPF1 RIP−Seq)

Recent progress in the technology of transcriptome-wide high-throughput sequencing has revealed that nonsense-mediated mRNA decay (NMD) targets ~10% of physiologic transcripts for the purpose of tuning gene expression in response to various environmental conditions. Regardless of the eukaryote studied, NMD requires the ATP-dependent RNA helicase upframeshift 1 (UPF1). It was initially thought that cellular NMD targets could be defined by their binding to steady-state UPF1, which is largely hypophosphorylated. However, the propensity for steady-state UPF1 to bind RNA nonspecifically, coupled with regulated phosphorylation of UPF1 on an NMD target serving as the trigger for NMD, made it clear that it is phosphorylated UPF1 (p-UPF1), rather than steady-state UPF1, that can be used to distinguish cellular NMD targets from cellular RNAs that are not. Here, we describe the immunoprecipitation of p-UPF1 followed by RNA sequencing (p-UPF1 RIP-seq) as a transcriptome-wide approach to define physiologic NMD targets.

Long-range PCR for the diagnosis of spinocerebellar ataxia type 10

Sirs, Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant neurodegenerative disorder characterized by a unique combination of progressive ataxia and seizures. The mutation causing SCA10 is an unstable and massive expansion (800∼4500 repeats; unaffected range, 10–29) of an ATTCT pentanucleotide repeat in intron 9 of the ataxin10 (ATXN10) gene on chromosome 22q13.3 [1]. In molecular analysis of SCA10, polymerase chain reaction (PCR) of the ATTCT repeat typically shows a single normal-sized allele since the expanded allele is nonamplifiable. Southern blot analysis is required to detect the repeat expansion and determine its size [1]. However, this method is laborious, costly, and not applicable to degraded samples or those with limited quantities of DNA. Ataxia patients are not often tested for SCA10 due to these limitations as well as insurance problems; a simple and cost-effective screening test would be preferred. Recent development of repeat-primed PCR (RP) [2, 3] allows quick detection of the ATTCT expansion by PCR alone, even in degraded DNA samples; however, it cannot determine the repeat length, merely the presence of expansion. Long-range PCR (LP) is effective for accurately and directly determining the size of expansions, requires little DNA, and can be carried out quickly. We recently succeeded in amplifying a 280-ATTCT expansion by LP [4]. Here, we show the improved LP protocol to accurately detect the full expansion ∼4,000 repeats, which can completely replace the use of Southern blot analysis. We used 100 normal controls and 20 patient-DNA samples in which the SCA10 diagnosis was confirmed and repeat sizes were determined by Southern analysis. LP was performed in the supplementary conditions. As shown in Fig. 1, we could detect variably expanded alleles ranging from 280 to 3,500 repeats. There were no false-positive results, i.e., no expansions in the 100 normal controls. A size estimate for each mutation was consistent with the results from Southern data, excluding the possibility of PCR artifact. We believe this protocol is a rapid and accurate method to both confirm the presence of an expansion mutation and determine its size without limitation of the mutation range, and samples positive for expansions by RP should be subjected to LP. Wick et al. [5] reported similar efforts in the diagnosis of Friedreich ataxia and established a protocol to amplify ∼1,700 GAA repeats by LP. Recent improvements in LP technology such as hot start and high-fidelity Pfu polymerase mixtures with 3′→5′proofreading activities are now allowing us to amplify longer, more complex genomic targets that were previously difficult to amplify. Our protocol will reduce time, labor, and cost requirements in clinical laboratory settings. It is superior to RP in molecular testing of SCA10 when the sizing of the expansion is critical (e.g., a family with reduced penetrance [4]) or homozygotes must be distinguished from heterozygotes. It will be also applicable to cloning of the expanded allele, Neurogenetics (2008) 9:151–152 DOI 10.1007/s10048-007-0117-x

Molecular autopsy provides evidence for widespread ribosome-phased mRNA fragmentation

Recent developments in transcriptome-wide sequencing technologies have enabled the identification of cellular mRNA decay intermediates. Although canonical mRNA decay has been shown to occur by deadenylation followed by decapping and subsequent exonucleolytic decay from both mRNA ends, a study by Mourelatos and colleagues now defines mRNA fragments that are generated on polysomes by endonucleolytic cleavages phased by the associated ribosome.