We have demonstrated the existence of multiple mRNA binding proteins that interact specifically with defined regions in posttranscriptionally regulated mRNAs. These domains appear to be destabilizers whose function can be attenuated by the interaction with the specific binding proteins. Thus, the ability to alter mRNA decay rates on demand, given different environmental or intracellular conditions, appears to be mediated by controlling the localization, activity, and overall function of the cognate binding protein. Based on our limited experience, we predict that most, if not all, of similarly regulated mRNAs will ultimately be found to interact with regulatory mRNA binding proteins. Under conditions whereby the mRNA binding proteins are constitutively active (e.g., tumor cell lines), abnormal mRNA decay will result, with accumulation and overtranslation. Such appears to be the case for cytokines and possibly amyloid protein precursor mRNAs in cancer and Alzheimer's disease, respectively. Conversely, mutagenesis of these critical 3' untranslated region elements will likely have comparable deleterious effects on the regulation of gene expression. To the extent that such derangements exist in human disease, attention to understanding the mechanistic detail at this level may provide insights into the development of appropriate therapeutics or treatment strategies.
"It is noteworthy to consider that there is not a direct correlation between mRNA transcripts and protein levels. Gene expression is also regulated by the control of mRNA degradation, since the steady-state concentration of mRNA is determined both by its rates of synthesis and decay (Rajagopalan and Malter 1997; Meyer et al. 2004). Changes in mRNA half-life do not reflect changes in transcription (Ross 1996). "
[Show abstract][Hide abstract] ABSTRACT: Autistic disorders (ADs) are heterogeneous neurodevelopmental disorders arised by the interaction of genes and environmental factors. Dysfunctions in social interaction and communication skills, repetitive and stereotypic verbal and non-verbal behaviours are common features of ADs. There are no defined mechanisms of pathogenesis, rendering curative therapy very difficult. Indeed, the treatments for autism presently available can be divided into behavioural, nutritional and medical approaches, although no defined standard approach exists. Autistic children display immune system dysregulation and show an altered immune response of peripheral blood mononuclear cells (PBMCs). In this study, we investigated the involvement of cannabinoid system in PBMCs from autistic children compared to age-matched normal healthy developing controls (age ranging 3-9 years; mean age: 6.06 ± 1.52 vs. 6.14 ± 1.39 in autistic children and healthy subjects, respectively). The mRNA level for cannabinoid receptor type 2 (CB2) was significantly increased in AD-PBMCs as compared to healthy subjects (mean ± SE of arbitrary units: 0.34 ± 0.03 vs. 0.23 ± 0.02 in autistic children and healthy subjects, respectively), whereas CB1 and fatty acid amide hydrolase mRNA levels were unchanged. mRNA levels of N-acylphosphatidylethanolamine-hydrolyzing phospholipase D gene were slightly decreased. Protein levels of CB-2 were also significantly increased in autistic children (mean ± SE of arbitrary units: 33.5 ± 1.32 vs. 6.70 ± 1.25 in autistic children and healthy subjects, respectively). Our data indicate CB2 receptor as potential therapeutic target for the pharmacological management of the autism care.
Journal of Autism and Developmental Disorders 04/2013; 43(11). DOI:10.1007/s10803-013-1824-9 · 3.06 Impact Factor
"The use of an in vitro protocol under highly controlled and reproducible conditions is warranted since our preliminary work revealed that measuring the stability of transcripts in skeletal muscle of living animals using transcription inhibitors such as actinomycin D, is unreliable and fraught with difficulties (data not shown). In fact, limitations of this approach have been discussed previously (34,36) and they include: (i) the toxicity of the drug which kills the animals in a few hours making extrapolations of half-life of relatively stable mRNAs questionable and (ii) the direct effect of these drugs on the mRNA decay process which may artificially alter the rate of degradation of specific cellular transcripts. "
[Show abstract][Hide abstract] ABSTRACT: We examined the role of post-transcriptional mechanisms in controlling utrophin A mRNA expression in slow versus fast skeletal muscles. First, we determined that the half-life of utrophin A mRNA is significantly shorter in the presence of proteins isolated from fast muscles. Direct plasmid injection experiments using reporter constructs containing the full-length or truncated variants of the utrophin 3'UTR into slow soleus and fast extensor digitorum longus muscles revealed that a region of 265 nucleotides is sufficient to confer lower levels of reporter mRNA in fast muscles. Further analysis of this region uncovered a conserved AU-rich element (ARE) that suppresses expression of reporter mRNAs in cultured muscle cells. Moreover, stability of reporter mRNAs fused to the utrophin full-length 3'UTR was lower in the presence of fast muscle protein extracts. This destabilization effect seen in vivo was lost upon deletion of the conserved ARE. Finally, we observed that calcineurin signaling affects utrophin A mRNA stability through the conserved ARE. These results indicate that ARE-mediated mRNA decay is a key mechanism that regulates expression of utrophin A mRNA in slow muscle fibers. This is the first demonstration of ARE-mediated mRNA decay regulating the expression of a gene associated with the slow myogenic program.
Nucleic Acids Research 03/2008; 36(3):826-38. DOI:10.1093/nar/gkm1107 · 9.11 Impact Factor
"However, the potential hairpin loop sequence was not present in all reported mammalian -subunit mRNA sequences; a G in the horse 3 UTR removes a potential base pair in the hairpin stem reducing the predicted G value to 1·0 kcal/mol, while the rat and mouse transcripts entirely lack the sequence (Chin et al. 1981, Godine et al. 1982). Conservation of mRNA secondary structure may indicate functional significance, and a role for hairpin loop structures in regulating mRNA stability in some transcripts is well established (Rajagopalan & Malter 1997). It is noteworthy that the position of the possum -subunit putative hairpin loop with respect to the translation termination codon was also conserved, varying between 177 and 188 nucleotides in the six -subunit 3 UTR sequences shown in Fig. 3. Furthermore, in five of the six 3 UTR sequences in Fig. 3, the putative hairpin sequence was located six or eight nucleotides 5 to the sequence AUUUA (AUUUUA in horse) which itself was positioned approximately 30 nucleotides 5 to the polyadenylation signal AAUAAA. "
[Show abstract][Hide abstract] ABSTRACT: A cDNA sequence from the gonadotrophin alpha-subunit mRNA of Australian brushtail possum (Trichosurus vulpecula) has been determined and analysed. Comparison with seven eutherian mammalian gonadotrophin alpha-subunit gene sequences revealed an average of 82.6% homology between the coding region nucleotide sequences and 88.8% identity between the predicted amino acid sequences. The predicted possum gonadotrophin alpha-subunit protein has ten evolutionarily conserved cysteine residues, two potential N-linked glycosylation sites and a putative enzyme recognition sequence which it has been suggested is required for sulphation of carbohydrate moieties. Comparison of the possum gonadotrophin alpha-subunit 3' untranslated region (UTR) sequence with the 3' UTRs of eutherian alpha-subunit transcripts revealed sequence homology. In particular, an 18 nucleotide imperfect palindromic sequence present in the possum 3' UTR, with the potential to form a hairpin loop, was found to be evolutionarily conserved and present in five out of seven eutherian alpha-subunit 3' UTR sequences. In situ hybridization localized the transcripts to a sub-population of anterior pituitary cells presumed to be gonadotrophs and thyrotrophs. In summary, these results indicate considerable conservation of the structure and function of the gonadotrophin alpha-subunit protein since the divergence of the marsupial and eutherian mammalian lineages.
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