Site-specific deoxynucleotide substitutions in yeast U6 snRNA block splicing of pre-mRNA in vitro.
We have identified 2'-hydroxyl groups of the U6 phosphate-ribose backbone which are required for reconstitution of splicing activity in U6-depleted yeast extract. To screen the 2'-hydroxyls of yeast U6 at nucleotides 39-88, spanning the conserved central domain, synthetic U6 RNAs were constructed with deoxyribonucleotides incorporated site specifically. Only four individual deoxynucleotide substitutions blocked splicing activity: dA51 (in the ACAGAG sequence), dA62 (next to the AGC triad), and dU70 and dC72 (both in the loop of the 3' intramolecular stem-loop). Native gel analysis revealed that these deoxy-substituted U6 RNAs were competent for assembly of spliceosomes. Interestingly, a 2'-O-methyl substituent at A51, A62, U70 or C72 did not inhibit splicing activity, indicating that the essential 2'-OH groups at these positions in U6 act as hydrogen bond acceptors or neutral coordinated ligands. The requisite 2'-hydroxyls at A62, U70 and C72 show both similarities and differences relative to the positions of essential 2'-hydroxyls of catalytic domain V of group II ribozymes. The identification of the essential 2'-hydroxyls at positions 62, 70 and 72 corroborates that the 3' intramolecular stem-loop in U6 plays an important role in pre-mRNA splicing.
Kim
C H
CH
Division of Biology, California Institute of Technology, Pasadena 91125, USA.
Ryan
D E
DE
Marciniec
T
T
Abelson
J
J
D001483:Base Sequence; D003854:Deoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry*; D017665:Hydroxyl Radical; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing / Q000502:physiology*; D012342:RNA, Small Nuclear / Q000737:chemistry* / Q000378:metabolism; D017381:Spliceosomes / Q000737:chemistry; D015003:Yeasts / Q000378:metabolism
D003854:Deoxyribonucleotides; D012322:RNA Precursors; D012342:RNA, Small Nuclear; D017665:Hydroxyl Radical