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BACKGROUND. Spinal muscular atrophy (SMA) is caused by deficient expression of survival motor neuron (SMN) protein. New SMN-enhancing therapeutics are associated with variable clinical benefits. Limited knowledge of baseline and drug-induced SMN levels in disease-relevant tissues hinders efforts to optimize these treatments. METHODS. SMN mRNA and protein levels were quantified in human tissues isolated during expedited autopsies. RESULTS. SMN protein expression varied broadly among prenatal control spinal cord samples, but was restricted at relatively low levels in controls and SMA patients after 3 months of life. A 2.3-fold perinatal decrease in median SMN protein levels was not paralleled by comparable changes in SMN mRNA. In tissues isolated from nusinersen-treated SMA patients, antisense oligonucleotide (ASO) concentration and full-length (exon 7 including) SMN2 (SMN2-FL) mRNA level increases were highest in lumbar and thoracic spinal cord. An increased number of cells showed SMN immunolabeling in spinal cord of treated patients, but was not associated with an increase in whole-tissue SMN protein levels. CONCLUSIONS. A normally occurring perinatal decrease in whole-tissue SMN protein levels supports efforts to initiate SMN-inducing therapies as soon after birth as possible. Limited ASO distribution to rostral spinal and brain regions in some patients likely limits clinical response of motor units in these regions for those patients. These results have important implications for optimizing treatment of SMA patients and warrant further investigations to enhance bioavailability of intrathecally administered ASOs. FUNDING. SMA Foundation, SMART, NIH (R01-NS096770, R01-NS062869), Ionis Pharmaceuticals, and PTC Therapeutics. Biogen provided support for absolute real-time RT-PCR., Introduction The neuromuscular disease spinal muscular atrophy (SMA), affecting approximately 1 in 10,000 individuals, is the most common inherited cause of infant death (1). The most frequent and severe type [...]

Nonsense (premature stop codon) mutations are causative in 5% to 15% of patients with monogenetic inherited disorders. PTC124, a 284-Dalton 1,2,4-oxadiazole, promotes ribosomal readthrough of premature stop codons in mRNA [...]

Dimethylation at arginine residues has been shown to be central in cellular processes such as signal transduction, transcription activation, and protein sorting. The two methyl groups are either placed symmetric or asymmetric on the [zeta] standing nitrogen atoms of the arginine side chain. Here, we introduce a novel method that enables the localization of dimethylarginine (DMA) residues in gel-separated proteins at a level of sensitivity of better than 1 pmol and that allows one to distinguish between the isomeric symmetric and asymmetric position of the methyl groups. The method utilizes two side-chain fragments of DMA, the dimethylammonium ion (m/z 46.06) and the dimethylcarbodiimidium ion (m/z 71.06), for positive ion mode precursor ion scanning. Dimethylcarbodiimidium ions (m/z 71.06) are produced by symmetric as well as asymmetric dimethylarginine but are observed more strongly for symmetric DMA. It is utilized here in the precursor of m/z 71 scan to indicate the presence of DMA in a peptide. The dimethylammonium ion (m/z 46.06) is specific for asymmetric DMA and is utilized here in the precursor of m/z 46 scan. The positive ion mode allows for the identification of the protein by peptide sequencing and simultaneous detection and localization of the modified residues. The analysis can be conducted on any mass spectrometer capable of precursor ion scanning. However, the high resolution of a quadrupole TOF instrument is beneficial to assign the accurate charge state of the often highly charged precursors. Using the precursor of m/z 71 scan, we found FUS/TLS and Sam68 to be DMA-containing proteins. We discovered at least 20 DMA sites in FUS/TLS. In MS/MS, we observed neutral loss of dimethylamine (m/z 45.05) from which it follows that the dimethylation in FUS/TLS is asymmetric. Monitoring in parallel the fragments m/z 46.06 and 71.06 in precursor ion scans and peptide sequencing, we identified at least nine asymmetric DMA modifications in Sam68. The parallel monitoring of fragments in precursor ion scans is a versatile tool to specify the nature of protein modifications in cases where a single fragment is not conclusive.

Research has ben conducted on the ribonucleoprotein (RNP) complexes. The identification of the RNP which sediments on surcose gradients and contains Germin3, Germin4 and human eIF2C2 along with small cellular RNAs has been carried out and the results of the genomic sequence analysis suggest that these small RNAs are derived from the larger precursors forming stem-loop structures.

The interaction of Upf1p and peptidyl release factors is critical for the putative surveillance complex formation and the degradation of aberrant mRNAs. It also improves translation termination and ensures that there is no premature translation. This nonsense-mediated mRNA decay pathway enables the cell to dispose of meaningless mutations.

A study of the identification and characterization of the human tRNA splicing endonuclease, a fundamental process required for cell growth and division along with an alternatively spliced isoform of SEN2, which is part of the RNA endonuclease activity is presented. The findings show that human endonuclease complexes are associated with pre-mRNA 3' end processing factors, and pre-tRNA splicing suggests that endonuclease subunits function in multiple RNA-processing events.

Background: The universal presence of a gene (SMN2) nearly identical to the mutated SMN1 gene responsible for Spinal Muscular Atrophy (SMA) has proved an enticing incentive to therapeutics development. Early disappointments from putative SMN-enhancing agent clinical trials have increased interest in improving the assessment of SMN expression in blood as an early "biomarker" of treatment effect. Methods: A cross-sectional, single visit, multi-center design assessed SMN transcript and protein in 108 SMA and 22 age and gender-matched healthy control subjects, while motor function was assessed by the Modified Hammersmith Functional Motor Scale (MHFMS). Enrollment selectively targeted a broad range of SMA subjects that would permit maximum power to distinguish the relative influence of SMN2 copy number, SMA type, present motor function, and age. Results: SMN2 copy number and levels of full-length SMN2 transcripts correlated with SMA type, and like SMN protein levels, were lower in SMA subjects compared to controls. No measure of SMN expression correlated strongly with MHFMS. A key finding is that SMN2 copy number, levels of transcript and protein showed no correlation with each other. Conclusion: This is a prospective study that uses the most advanced techniques of SMN transcript and protein measurement in a large selectively-recruited cohort of individuals with SMA. There is a relationship between measures of SMN expression in blood and SMA type, but not a strong correlation to motor function as measured by the MHFMS. Low SMN transcript and protein levels in the SMA subjects relative to controls suggest that these measures of SMN in accessible tissues may be amenable to an "early look" for target engagement in clinical trials of putative SMN-enhancing agents. Full length SMN transcript abundance may provide insight into the molecular mechanism of phenotypic variation as a function of SMN2 copy number. [ABSTRACT FROM AUTHOR]

Background: Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRFl and eRF3. eRFl recognizes nonsense codons UAA, UAG and UGA, while eRF3 stimulates polypeptide release from the ribosome in a GTP- and eRFl -- dependent manner. Recent studies has shown that proteins interacting with these release factors can modulate the efficiency of nonsense codon readthrough. Results: We have isolated a nonessential yeast gene, which causes suppression of nonsense mutations, being in a multicopy state. This gene encodes a protein designated Itt1p, possessing a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes. Overexpression of Itt1p decreases the efficiency of translation termination, resulting in the readthrough of all three types of nonsense codons. Itt1p interacts in vitro with both eRFl and eRF3. Overexpression of eRFl, but not of eRF3, abolishes the nonsense suppressor effect of overexpressed Itt1p. Conclusions: The data obtained demonstrate that Itt1p can modulate the efficiency of translation termination in yeast. This protein possesses a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes, and this is a first observation of such protein being involved in translation. [ABSTRACT FROM AUTHOR]

The SUP35 gene of Saccharomyces cerevisiae encodes the polypeptide chain release factor eRF3. This protein (also called Sup35p) is thought to be able to undergo a heritable conformational switch, similarly to mammalian prions, giving rise to the cytoplasmically inherited ψ+ determinant. A dominant mutation (PNM2 allele) in the SUP35 gene causing a Gly58→Asp change in the Sup35p N-terminal domain eliminates ψ+. Here we observed that the mutant Sup35p can be converted to the prion­like form in vitro, but such conversion proceeds slower than that of wild-type Sup35p. The overexpression of mutant Sup35p induced the de novo appearance of ψ+ cells containing the prion­like form of mutant Sup35p, which was able to transmit its properties to wild-type Sup35p both in vitro and in vivo. Our data indicate that this ψ+-eliminating mutation does not alter the initial binding of Sup35p molecules to the Sup35p ψ+-specific aggregates, but rather inhibits its subsequent prion­like rearrangement and/or binding of the next Sup35p molecule to the growing prion­like Sup35p aggregate. [ABSTRACT FROM AUTHOR]

Spinal muscular atrophy (SMA) is caused by the loss or mutation of both copies of the survival motor neuron 1 (SMN1) gene. The related SMN2 gene is retained, but due to alternative splicing of exon 7, produces insufficient levels of the SMN protein. Here, we systematically characterize the pharmacokinetic and pharmacodynamics properties of the SMN splicing modifier SMN-C1. SMN-C1 is a low-molecular weight compound that promotes the inclusion of exon 7 and increases production of SMN protein in human cells and in two transgenic mouse models of SMA. Furthermore, increases in SMN protein levels in peripheral blood mononuclear cells and skin correlate with those in the central nervous system (CNS), indicating that a change of these levels in blood or skin can be used as a non-invasive surrogate to monitor increases of SMN protein levels in the CNS. Consistent with restored SMN function, SMN-C1 treatment increases the levels of spliceosomal and U7 small-nuclear RNAs and corrects RNA processing defects induced by SMN deficiency in the spinal cord of SMNΔ7 SMA mice. A 100% or greater increase in SMN protein in the CNS of SMNΔ7 SMA mice robustly improves the phenotype. Importantly, a ∼50% increase in SMN leads to long-term survival, but the SMA phenotype is only partially corrected, indicating that certain SMA disease manifestations may respond to treatment at lower doses. Overall, we provide important insights for the translation of pre-clinical data to the clinic and further therapeutic development of this series of molecules for SMA treatment., (© The Author 2016. Published by Oxford University Press.)