Antisense probes containing 2-aminoadenosine allow efficient depletion of U5 snRNP from HeLa splicing extracts.
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.
Lamm
G M
GM
EMBL, Heidelberg, FRG.
Blencowe
B J
BJ
Sproat
B S
BS
Iribarren
A M
AM
Ryder
U
U
Lamond
A I
AI
D000241:Adenosine / Q000031:analogs & derivatives* / Q000737:chemistry; D001483:Base Sequence; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000378:metabolism; D015347:RNA Probes / Q000737:chemistry / Q000378:metabolism*; D012326:RNA Splicing / Q000502:physiology*; D016372:RNA, Antisense / Q000737:chemistry / Q000378:metabolism*; D016914:Ribonuclease H; D012261:Ribonucleoproteins / Q000378:metabolism / Q000502:physiology*; D017411:Ribonucleoproteins, Small Nuclear
D012322:RNA Precursors; D015347:RNA Probes; D016372:RNA, Antisense; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; C033854:2-aminoadenosine; D004247:DNA; D004722:Endoribonucleases; D016914:Ribonuclease H; D000241:Adenosine