Point mutations in the stem-loop at the 3' end of mouse histone mRNA reduce expression by reducing the efficiency of 3' end formation.
Mammalian histone mRNAs end in a highly conserved stem-loop structure, with a six-base stem and a four-base loop. We have examined the effect of mutating the stem-loop on the expression of the histone mRNA in vivo by introducing the mutated histone genes into CHO cells by stable transfection. Point mutations have been introduced into the loop sequence and into the UA base pair at the top of the stem. Changing either the first or the third base of the conserved UYUN sequence in the loop to a purine greatly reduced expression, while changing both U's to purines abolished expression. A number of alterations in the stem sequence, including reversing the stem sequence, reversing the two base pairs at the base of the stem, or destroying the UA base pair at the top of the stem, also abolished expression. Changing the UA base pair to a CG or a UG base pair also reduced expression. The loss of expression is due to inefficient processing of the pre-mRNA, as judged by the efficiency of processing in vitro. Addition of a polyadenylation site or the wild-type histone processing signal downstream of a mutant stem-loop resulted in rescuing the processing of the mutant pre-histone mRNA. These results suggest that if the histone pre-mRNA is not rapidly processed, then it is degraded.
Pandey
N B
NB
Institute of Molecular Biophysics, Florida State University, Tallahassee 32306.
Williams
A S
AS
Sun
J H
JH
Brown
V D
VD
Bond
U
U
Marzluff
W F
WF
D000818:Animals; D001483:Base Sequence; D015870:Gene Expression; D006367:HeLa Cells; D006657:Histones / Q000235:genetics*; D006801:Humans; D006860:Hydrogen Bonding; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D016601:RNA-Binding Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D006657:Histones; D012333:RNA, Messenger; D016601:RNA-Binding Proteins