Antisense probes containing 2-aminoadenosine allow efficient depletion of U5 snRNP from HeLa splicing extracts.

AUTOR(ES)

Lamm, G M

RESUMO

RNA duplexes containing the modified base 2-amino-adenine in place of adenine are stabilized through the formation of three hydrogen bonds in 2-amino A.U base pairs. Antisense 2'-O-alkyloligoribonucleotide probes incorporating 2-aminoadenosine are thus able to efficiently affinity select RNP particles which are otherwise inaccessible. This has allowed the efficient and specific depletion of U5 snRNP from HeLa cell nuclear splicing extracts. U5 snRNP is shown to be essential for spliceosome assembly and for both steps of pre-mRNA splicing. The absence of U5 snRNP prevents the stable association of U4/U6 but not U1 and U2 snRNPs with pre-mRNA.

Documentos Relacionados

• Antisense oligonucleotide binding to U5 snRNP induces a conformational change that exposes the conserved loop of U5 snRNA.
• Genetic depletion indicates a late role for U5 snRNP during in vitro spliceosome assembly.
• Evidence from complementation assays in vitro that U5 snRNP is required for both steps of mRNA splicing.
• Antisense probes targeted to an internal domain in U2 snRNP specifically inhibit the second step of pre-mRNA splicing.
• Purification and characterization of a simple ribonucleoprotein particle containing small nucleoplasmic RNAs (snRNP) as a subset of RNP containing heterogenous nuclear RNA (hnRNP) from HeLa cells.