Characterization of the cis-acting element directing perinuclear localization of the metallothionein-1 mRNA.

Subcellular localization of mRNAs is a key mechanism for the synthesis of proteins close to their site of function. The mRNA encoding MT-1 (metallothionein-1) is localized in the perinuclear cytoplasm, where it is associated with cytoskeletal-bound polysomes. This localization relies on sequences present in the 3'-UTR (3'-untranslated region). The present study aims to characterize the cis-acting localization element(s) within the 3'-UTR. Using transfected cells expressing tagged MT-1 differing in their 3'-UTRs (deleted or mutated), the section(s) of this region required for directing MT-1 transcripts to the perinuclear cytoplasm has been investigated. Different 3'-UTRs were also used in UV cross-linking experiments that highlighted two distinct regions (nt 26-30 and 66-76) necessary for the binding of a protein of approx. 50 kDa, presumably involved in the mRNA targeting. The poor sequence homology between the MT-1 3'-UTR of various species, together with the bipartite nature of the required cis-element, indicates the involvement of a particular structure in the localization signal. The secondary structure of the MT-1 3'-UTR was investigated using enzymic and chemical probing. Current structural analysis of mutant 3'-UTRs will allow the critical structural features of the MT-1 mRNA perinuclear localization signal to be defined.

Isolation and identification of a protein binding to the localization element of Metallothionein-1 mRNA.

mRNA localization provides a mechanism for localized protein synthesis. mRNAs encoding certain proteins, including c-MYC, c-FOS, MT-1 (Metallothionein-1) and vimentin, are localized around the nuclei of mammalian cells and are associated with the cytoskeleton. Targeting of these mRNAs to the perinuclear cytoplasm is mediated by elements within their 3'-UTRs (3'-untranslated regions), but many of the trans-acting proteins remain unidentified. UV cross-linking assays using radiolabelled transcripts indicated that a protein of approx. 50 kDa (from the Chinese-hamster ovary cell extracts) bound to the MT-1 3'-UTR sequence. Competition experiments using unlabelled mutant 3'-UTR RNAs revealed that the binding of this protein is specific to localization-positive mutants. Isolation of a 50 kDa protein was achieved by an RNA affinity-based method in which biotinylated MT-1 3'-UTR RNA was anchored to paramagnetic beads. Bound proteins were eluted and analysed by SDS/PAGE. The 50 kDa protein was extracted from the gel, subjected to trypsin digestion and identified by matrix-assisted laser-desorption/ionization-time-of-flight mass spectrometry as eukaryote elongation factor 1alpha.

Susceptibility markers in colorectal cancer.

Many susceptibility factors contribute to an individual's risk of developing colorectal cancer. Family history of colorectal cancer (particularly with early age of onset), maleness and increasing age are all factors associated with increasing risk. About three quarters of colorectal cancers are thought to be due to somatic mutations, and both high- and low-penetrance predisposing genes contribute to the remaining quarter of cases. Many of the highly penetrant dominant genes are known, but others remain to be identified. Describing the contribution of individual genes is likely to be very complex, as some modify the impact of other genes and other environmental factors rather than incurring a direct, easily attributable effect. The two dominant predisposing syndromes are familial adenomatous polyposis and Lynch syndrome, the first due to a mutant tumour-suppressor gene APC, and the second due to mutations in a number of genes responsible for mismatch repair in DNA at cell division. Establishing genetic susceptibility for colorectal cancer will soon be possible, and could save lives by allowing targetting of screening and the encouragement of preventive behaviours. However, there will always be a risk of making healthy people "sick" through the identification of predisposing genes, and there are many potential ways by which a gene carrier may be stigmatized by society, insurance companies and employers.