Itt1p, a novel protein inhibiting translation termination in Saccharomyces cerevisiae

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

Evaluation of SMN Protein, Transcript, and Copy Number in the Biomarkers for Spinal Muscular Atrophy (BforSMA) Clinical Study

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. TRIAL REGISTRY: Clinicaltrials.gov NCT00756821

Normalization of Patient-Identified Plasma Biomarkers in SMNΔ7 Mice following Postnatal SMN Restoration

<div><p>Introduction and Objective</p><p>Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disorder. SMA is caused by homozygous loss of the <i>SMN1</i> gene and retention of the <i>SMN2</i> gene resulting in reduced levels of full length SMN protein that are insufficient for motor neuron function. Various treatments that restore levels of SMN are currently in clinical trials and biomarkers are needed to determine the response to treatment. Here, we sought to investigate in SMA mice a set of plasma analytes, previously identified in patients with SMA to correlate with motor function. The goal was to determine whether levels of plasma markers were altered in the SMNΔ7 mouse model of SMA and whether postnatal SMN restoration resulted in normalization of the biomarkers.</p><p>Methods</p><p>SMNΔ7 and control mice were treated with antisense oligonucleotides (ASO) targeting ISS-N1 to increase SMN protein from <i>SMN2</i> or scramble ASO (sham treatment) via intracerebroventricular injection on postnatal day 1 (P1). Brain, spinal cord, quadriceps muscle, and liver were analyzed for SMN protein levels at P12 and P90. Ten plasma biomarkers (a subset of biomarkers in the SMA-MAP panel available for analysis in mice) were analyzed in plasma obtained at P12, P30, and P90.</p><p>Results</p><p>Of the eight plasma biomarkers assessed, 5 were significantly changed in sham treated SMNΔ7 mice compared to control mice and were normalized in SMNΔ7 mice treated with ASO.</p><p>Conclusion</p><p>This study defines a subset of the SMA-MAP plasma biomarker panel that is abnormal in the most commonly used mouse model of SMA. Furthermore, some of these markers are responsive to postnatal SMN restoration. These findings support continued clinical development of these potential prognostic and pharmacodynamic biomarkers.</p></div

Tissue homogenization in ER4 lysis buffer and dilutions for protein and ELISA assays.

<p>Tissue homogenization in ER4 lysis buffer and dilutions for protein and ELISA assays.</p

Longitudinal electrophysiology.

<p>At P12 CMAP amplitude is reduced in ASO-SMA mice (n = 5) compared with ASO-Het (n = 8) (p = 0.045). Otherwise there are no statistically significant differences. Compound muscle action potential, CMAP; motor unit number estimation, MUNE; and average Single Motor Unit Potential, SMUP.</p

Whole blood SMN levels at P12, P30, and P90.

<p>SMN levels were between ASO-treated SMA mice and untreated SMA, ASO-treated Het mice, and untreated Het mice. At P12, SMN levels were increased in ASO-SMA mice (n = 11) compared to SMA mice (n = 13) but reduced compared to ASO-Het (n = 10) and Het mice (n = 4). Untreated SMA mice have a mean survival of ~2 weeks. Therefore no SMA disease control animals are available for comparison at P30 or P90. At P30 SMN levels were diminished in ASO-SMA mice (n = 10) compared to ASO-Het (n = 5) and Het (n = 8) mice. At P90, SMN levels in ASO-SMA mice (n = 11) were reduced compared with ASO-Het (n = 6) but not Het (n = 8) mice. *, p<0.05, ***, p<0.001 (Note the different scale for P12, P30, and P90 SMN levels).</p

Biomarkers in SMA Mice, SMN-restored Mice, and Human SMA.

<p>Biomarkers in SMA Mice, SMN-restored Mice, and Human SMA.</p

Analytes that are not responsive to SMN.

<p>ASO-Het mice were considered controls for statistical comparison using One-way ANOVA and Dunnett's multiple comparisons test (GraphPad Prism, La Jolla CA). COMP was significantly reduced in ASO-SMA (n = 12) and SMA (n = 13) compared with ASO-Het (n = 10). There was no significant difference between Het (n = 5) and ASO-Het mice for COMP. AXL and CHI3LI were unchanged in all groups compared with ASO-Het mice. ** <0.01, *** <0.001.</p