Biomedical subjects

Andrew M MacMillan

Publications and source records attributed to Andrew M MacMillan.

6 recordsLinked to original sources

PubMed2006

FRET analysis of in vivo dimerization by RNA-editing enzymes.

Members of the ADAR (adenosine deaminase that acts on RNA) enzyme family catalyze the hydrolytic deamination of adenosine to inosine within double-stranded RNAs, a poorly understood process that is critical to mammalian development. We have performed fluorescence resonance energy transfer experiments in mammalian cells transfected with fluorophore-bearing ADAR1 and ADAR2 fusion proteins to investigate the relationship between these proteins. These studies conclusively demonstrate the homodimerization of ADAR1 and ADAR2 and also show that ADAR1 and ADAR2 form heterodimers in human cells. RNase treatment of cells expressing these fusion proteins changes their localization but does not affect dimerization. Taken together these results suggest that homo- and heterodimerization are important for the activity of ADAR family members in vivo and that these associations are RNA independent.

Adenosine Deaminase↗

PubMed2006

Crystal structure of a core spliceosomal protein interface.

The precise excision of introns from precursor mRNAs (pre-mRNAs) in eukaryotes is accomplished by the spliceosome, a complex assembly containing five small nuclear ribonucleoprotein (snRNP) particles. Human p14, a component of the spliceosomal U2 and U11/U12 snRNPs, has been shown to associate directly with the pre-mRNA branch adenosine early in spliceosome assembly and within the fully assembled spliceosome. Here we report the 2.5-A crystal structure of a complex containing p14 and a peptide derived from the p14-associated U2 snRNP component SF3b155. p14 contains an RNA recognition motif (RRM), the surface of which is largely occluded by a C-terminal alpha-helix and a portion of the SF3b155 peptide. An analysis of RNA.protein crosslinking to wild-type and mutant p14 shows that the branch adenosine directly interacts with a conserved aromatic within a pocket on the surface of the complex. This result, combined with a comparison of the structure with known RRMs and pseudoRRMs as well as model-building by using the electron cryomicroscopy structure of a spliceosomal U11/U12 di-snRNP, suggests that p14.SF3b155 presents a noncanonical surface for RNA recognition at the heart of the mammalian spliceosome.

Adenosine↗

PubMed2004

RNAi: running interference for the cell.

RNA interference or RNAi is a recently characterized mechanism of eukaryotic gene regulation in which a short sequence of double-stranded RNA (dsRNA) specifically down-regulates expression of the associated gene. Preliminary characterization of this phenomenon has revealed a set of inter-related cellular pathways which appear to represent both a response to foreign RNA and a mechanism of endogenous gene regulation. Introduction of dsRNA into cells by a variety of means, including transfection of synthetic RNA duplexes, triggers the RNAi response resulting in specific suppression of target gene expression. Recent efforts on a genome wide scale have involved application of RNAi as an important new tool in cell biology to elucidate gene function in living cells.

Animals↗

PubMed2003

Structuring of the 3' splice site by U2AF65.

Recognition of the 3' splice site in mammalian introns is accomplished by association of the splicing factor U2AF with the precursor mRNA (pre-mRNA) in a multiprotein splicing commitment complex. It is well established that this interaction involves binding of the large U2AF65 subunit to sequences upstream of the 3' splice site, but the orientation of the four domains of this protein with respect to the RNA and hence their role in structuring the commitment complex remain unclear and the basis of contradictory models. We have examined the interaction of U2AF65 with an RNA representing the 3' splice site using a series of U2AF deletion mutants modified at the N terminus with the directed hydroxyl radical probe iron-EDTA. These studies, combined with an analysis of extant high resolution x-ray structures of protein.RNA complexes, suggest a model whereby U2AF65 bends the pre-mRNA to juxtapose reactive functionalities of the pre-mRNA substrate and organize these structures for subsequent spliceosome assembly.

Binding Sites↗

PubMed2002

Adenosine to inosine editing by ADAR2 requires formation of a ternary complex on the GluR-B R/G site.

RNA editing by members of the ADAR (adenosine deaminase that acts on RNA) enzyme family involves hydrolytic deamination of adenosine to inosine within the context of a double-stranded pre-mRNA substrate. Editing of the human GluR-B transcript is catalyzed by the enzyme ADAR2 at the Q/R and R/G sites. We have established a minimal RNA substrate for editing based on the R/G site and have characterized the interaction of ADAR2 with this RNA by gel shift, kinetic, and cross-linking analyses. Gel shift analysis revealed that two complexes are formed on the RNA as protein concentration is increased; the ADAR monomers can be cross-linked to one another in an RNA-dependent fashion. We performed a detailed kinetic study of the editing reaction; the data from this study are consistent with a reaction scheme in which formation of an ADAR2.RNA ternary complex is required for efficient RNA editing and in which formation of this complex is rate determining. These observations suggest that RNA adenosine deaminases function as homodimers on their RNA substrates and may partially explain regulation of RNA editing in these systems.

Adenosine↗

PubMed2002

Early organization of pre-mRNA during spliceosome assembly.

Intron excision from precursor mRNAs (pre-mRNAs) in eukaryotes requires juxtaposition of reactive functionalities within the substrate at the heart of the spliceosome where the two chemical steps of splicing occur. Although a series of interactions between pre-mRNAs, pre-spliceosomal and spliceosomal factors is well established, the molecular mechanisms of splicing machinery assembly, as well as the temporal basis for organization of the substrate for splicing, remain poorly understood. Here we have used a directed hydroxyl radical probe tethered to pre-mRNA substrates to map the structure of the pre-mRNA substrate during the spliceosome assembly process. These studies indicate an early organization and proximation of conserved pre-mRNA sequences during spliceosome assembly/recruitment and suggest a mechanism for the formation of the final active site of the mature spliceosome.

Adenosine Triphosphate↗