Beyond the Identification of Transcribed Sequences: Functional and Expression Analysis

9th Annual Workshop, October 28-31, 1999

Co-sponsored by the U.S. Department of Energy


HESKETH

Perinuclear mRNA localisation by 3'untranslated sequences

John Hesketh1, Marilyne Levadoux1, Gillian Dalgleish1, John Beattie1, Heather Wallace2, and Jean-Marie Blanchard3

1Rowett Research Institute, Buckburn, Aberdeen, Scotland
2Dept of Medicine and Therapeutics, University of Aberdeen, United Kingdom
3IGMM-CNRS, Montpellier, France

There is increasing evidence that mRNAs can be found localised in different regions of the cytoplasm. Such mRNA localisation occurs in a variety of cells from yeast to mammals, as does the association of mRNAs with the cytoskeleton. Both association of mRNAs with the cytoskeleton and mRNA localisation depend in the majority of cases on signals within the 3' untranslated sequences (3'UTRs) of the mRNAs. Using cell fractionation techniques the mRNAs encoding c-myc, c-fos, ribosomal proteins L1 and S6, cyclin A and metallothionein (MT1) have been found associated with the cytoskeleton. These mRNAs all code for proteins which under all or some circumstances are imported into the nucleus after synthesis; e.g. MYC and FOS are transcription factors and MT1 can be found in the nucleus during

S-phase of the cell cycle. In situ hybridisation shows that c-myc and MT1mRNAs are localised around the nucleus. In addition analysis of mammalian cell lines expressing chimaeric gene constructs in which coding or reporter sequences are linked to different 3'UTRs shows that c-myc, c-fos and MT1 3'UTRs all contain sequences capable of targeting mRNAs to the perinuclear cytoplasm and the cytoskeleton.

Recently, we have developed clonal cell lines transfected with constructs which produce either normal localisation of MT1-encoding transcripts or delocalised transcripts. Using these cell lines it has been found that loss of localisation of MT1transcripts is associated with a lack of MT1 in the nucleus at the G1-S transition of the cell cycle.

These data lead to the hypothesis that 1] certain mRNAs are targeted to the perinuclear cytoplasm by signals in their 3'UTRs and that this functions to promote the efficient imported of the newly synthesised proteins into the nucleus, and 2] this mechanism is a general one utilised by a range of nuclear proteins and proteins which shuttle from cytoplasm to nucleus during the cell cycle. To identify other mRNAs which may be localised in this way the strategy is now to define the localisation signal required for c-myc and metallothionein mRNA localisation, to predict a concensus structure and then search databases to identify other mRNAs containing a similar signal.


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