A nascent peptide signal responsive to endogenous levels of polyamines acts to stimulate regulatory frameshifting on antizyme mRNA.

The protein antizyme is a negative regulator of cellular polyamine concentrations from yeast to mammals. Synthesis of functional antizyme requires programmed +1 ribosomal frameshifting at the 3' end of the first of two partially overlapping ORFs. The frameshift is the sensor and effector in an autoregulatory circuit. Except for Saccharomyces cerevisiae antizyme mRNA, the frameshift site alone only supports low levels of frameshifting. The high levels usually observed depend on the presence of cis-acting stimulatory elements located 5' and 3' of the frameshift site. Antizyme genes from different evolutionary branches have evolved different stimulatory elements. Prior and new multiple alignments of fungal antizyme mRNA sequences from the Agaricomycetes class of Basidiomycota show a distinct pattern of conservation 5' of the frameshift site consistent with a function at the amino acid level. As shown here when tested in Schizosaccharomyces pombe and mammalian HEK293T cells, the 5' part of this conserved sequence acts at the nascent peptide level to stimulate the frameshifting, without involving stalling detectable by toe-printing. However, the peptide is only part of the signal. The 3' part of the stimulator functions largely independently and acts at least mostly at the nucleotide level. When polyamine levels were varied, the stimulatory effect was seen to be especially responsive in the endogenous polyamine concentration range, and this effect may be more general. A conserved RNA secondary structure 3' of the frameshift site has weaker stimulatory and polyamine sensitizing effects on frameshifting

Translation initiation downstream from annotated start codons in human mRNAs coevolves with the Kozak context

Eukaryotic translation initiation involves preinitiation ribosomal complex 5′ -to-3′ directional probing of mRNA for codons suitable for starting protein synthesis. The recognition of codons as starts depends on the codon identity and on its immediate nucleotide context known as Kozak context. When the context is weak (i.e., nonoptimal), leaky scanning takes place during which a fraction of ribosomes continues the mRNA probing. We explored the relationship between the context of AUG codons annotated as starts of protein-coding sequences and the next AUG codon occurrence. We found that AUG codons downstream from weak starts occur in the same frame more frequently than downstream from strong starts. We suggest that evolutionary selection on in-frame AUGs downstream from weak start codons is driven by the advantage of the reduction of wasteful out-of-frame product synthesis and also by the advantage of producing multiple proteoforms from certain mRNAs. We confirmed translation initiation downstream from weak start codons using ribosome profiling data. We also tested translation of alternative start codons in 10 specific human genes using reporter constructs. In all tested cases, initiation at downstream start codons was more productive than at the annotated ones. In most cases, optimization of Kozak context did not completely abolish downstream initiation, and in the specific example of CMPK1 mRNA, the optimized start remained unproductive. Collectively, our work reveals previously uncharacterized forces shaping the evolution of protein-coding genes and points to the plurality of translation initiation and the existence of sequence features influencing start codon selection, other than Kozak context.Russian Science Foundation (RSF) 20-14-00121Science Foundation Ireland 210692/Z/18/ZScience Foundation Ireland 12/RC/2276_P2Erasmus+ ProgrammePlan Propio de Investigacion 2019 de la Universidad de GranadaMinistry of Economy of Spain DPI2017-84439-REuropean Union (EU) DPI2017-84439-

AMD1 mRNA employs ribosome stalling as a mechanism for molecular memory formation.

In addition to acting as template for protein synthesis, messenger RNA (mRNA) often contains sensory sequence elements that regulate this process1,2. Here we report a new mechanism that limits the number of complete protein molecules that can be synthesized from a single mRNA molecule of the human AMD1 gene encoding adenosylmethionine decarboxylase 1 (AdoMetDC). A small proportion of ribosomes translating AMD1 mRNA stochastically read through the stop codon of the main coding region. These readthrough ribosomes then stall close to the next in-frame stop codon, eventually forming a ribosome queue, the length of which is proportional to the number of AdoMetDC molecules that were synthesized from the same AMD1 mRNA. Once the entire spacer region between the two stop codons is filled with queueing ribosomes, the queue impinges upon the main AMD1 coding region halting its translation. Phylogenetic analysis suggests that this mechanism is highly conserved in vertebrates and existed in their common ancestor. We propose that this mechanism is used to count and limit the number of protein molecules that can be synthesized from a single mRNA template. It could serve to safeguard from dysregulated translation that may occur owing to errors in transcription or mRNA damage

A nascent peptide signal responsive to endogenous levels of polyamines acts to stimulate regulatory frameshifting on antizyme mRNA.

The protein antizyme is a negative regulator of cellular polyamine concentrations from yeast to mammals. Synthesis of functional antizyme requires programmed +1 ribosomal frameshifting at the 3\u27 end of the first of two partially overlapping ORFs. The frameshift is the sensor and effector in an autoregulatory circuit. Except for Saccharomyces cerevisiae antizyme mRNA, the frameshift site alone only supports low levels of frameshifting. The high levels usually observed depend on the presence of cis-acting stimulatory elements located 5\u27 and 3\u27 of the frameshift site. Antizyme genes from different evolutionary branches have evolved different stimulatory elements. Prior and new multiple alignments of fungal antizyme mRNA sequences from the Agaricomycetes class of Basidiomycota show a distinct pattern of conservation 5\u27 of the frameshift site consistent with a function at the amino acid level. As shown here when tested in Schizosaccharomyces pombe and mammalian HEK293T cells, the 5\u27 part of this conserved sequence acts at the nascent peptide level to stimulate the frameshifting, without involving stalling detectable by toe-printing. However, the peptide is only part of the signal. The 3\u27 part of the stimulator functions largely independently and acts at least mostly at the nucleotide level. When polyamine levels were varied, the stimulatory effect was seen to be especially responsive in the endogenous polyamine concentration range, and this effect may be more general. A conserved RNA secondary structure 3\u27 of the frameshift site has weaker stimulatory and polyamine sensitizing effects on frameshifting

Translation initiation downstream from annotated start codons in human mRNAs coevolves with the Kozak context

Eukaryotic translation initiation involves preinitiation ribosomal complex 5'-to-3' directional probing of mRNA for codons suitable for starting protein synthesis. The recognition of codons as starts depends on the codon identity and on its immediate nucleotide context known as Kozak context. When the context is weak (i.e., nonoptimal), leaky scanning takes place during which a fraction of ribosomes continues the mRNA probing. We explored the relationship between the context of AUG codons annotated as starts of protein-coding sequences and the next AUG codon occurrence. We found that AUG codons downstream from weak starts occur in the same frame more frequently than downstream from strong starts. We suggest that evolutionary selection on in-frame AUGs downstream from weak start codons is driven by the advantage of the reduction of wasteful out-of-frame product synthesis and also by the advantage of producing multiple proteoforms from certain mRNAs. We confirmed translation initiation downstream from weak start codons using ribosome profiling data. We also tested translation of alternative start codons in 10 specific human genes using reporter constructs. In all tested cases, initiation at downstream start codons was more productive than at the annotated ones. In most cases, optimization of Kozak context did not completely abolish downstream initiation, and in the specific example of CMPK1 mRNA, the optimized start remained unproductive. Collectively, our work reveals previously uncharacterized forces shaping the evolution of protein-coding genes and points to the plurality of translation initiation and the existence of sequence features influencing start codon selection, other than Kozak context

Heterogeneous genetic background of the association of pheochromocytoma/paraganglioma and pituitary adenoma: results from a large patient cohort

CONTEXT: Pituitary adenomas and pheochromocytomas/paragangliomas (pheo/PGL) can occur in the same patient or in the same family. Coexistence of the two diseases could be due to either a common pathogenic mechanism or a coincidence. OBJECTIVE: The objective of the investigation was to study the possible coexistence of pituitary adenoma and pheo/PGL. DESIGN: Thirty-nine cases of sporadic or familial pheo/PGL and pituitary adenomas were investigated. Known pheo/PGL genes (SDHA-D, SDHAF2, RET, VHL, TMEM127, MAX, FH) and pituitary adenoma genes (MEN1, AIP, CDKN1B) were sequenced using next generation or Sanger sequencing. Loss of heterozygosity study and pathological studies were performed on the available tumor samples. SETTING: The study was conducted at university hospitals. PATIENTS: Thirty-nine patients with sporadic of familial pituitary adenoma and pheo/PGL participated in the study. OUTCOME: Outcomes included genetic screening and clinical characteristics. RESULTS: Eleven germline mutations (five SDHB, one SDHC, one SDHD, two VHL, and two MEN1) and four variants of unknown significance (two SDHA, one SDHB, and one SDHAF2) were identified in the studied genes in our patient cohort. Tumor tissue analysis identified LOH at the SDHB locus in three pituitary adenomas and loss of heterozygosity at the MEN1 locus in two pheochromocytomas. All the pituitary adenomas of patients affected by SDHX alterations have a unique histological feature not previously described in this context. CONCLUSIONS: Mutations in the genes known to cause pheo/PGL can rarely be associated with pituitary adenomas, whereas mutation in a gene predisposing to pituitary adenomas (MEN1) can be associated with pheo/PGL. Our findings suggest that genetic testing should be considered in all patients or families with the constellation of pheo/PGL and a pituitary adenoma.This article is available via Open Access. Please click on the 'Additional Link' above to access the full-text

Chronic coronary syndromes without standard modifiable cardiovascular risk factors and outcomes: the CLARIFY registry

Background and Aims: It has been reported that patients without standard modifiable cardiovascular (CV) risk factors (SMuRFs—diabetes, dyslipidaemia, hypertension, and smoking) presenting with first myocardial infarction (MI), especially women, have a higher in-hospital mortality than patients with risk factors, and possibly a lower long-term risk provided they survive the post-infarct period. This study aims to explore the long-term outcomes of SMuRF-less patients with stable coronary artery disease (CAD). Methods: CLARIFY is an observational cohort of 32 703 outpatients with stable CAD enrolled between 2009 and 2010 in 45 countries. The baseline characteristics and clinical outcomes of patients with and without SMuRFs were compared. The primary outcome was a composite of 5-year CV death or non-fatal MI. Secondary outcomes were 5-year all-cause mortality and major adverse cardiovascular events (MACE—CV death, non-fatal MI, or non-fatal stroke). Results: Among 22 132 patients with complete risk factor and outcome information, 977 (4.4%) were SMuRF-less. Age, sex, and time since CAD diagnosis were similar across groups. SMuRF-less patients had a lower 5-year rate of CV death or non-fatal MI (5.43% [95% CI 4.08–7.19] vs. 7.68% [95% CI 7.30–8.08], P = 0.012), all-cause mortality, and MACE. Similar results were found after adjustments. Clinical event rates increased steadily with the number of SMuRFs. The benefit of SMuRF-less status was particularly pronounced in women. Conclusions: SMuRF-less patients with stable CAD have a substantial but significantly lower 5-year rate of CV death or non-fatal MI than patients with risk factors. The risk of CV outcomes increases steadily with the number of risk factors