The efficiency of 3'-end formation contributes to the relative levels of different histone mRNAs.

Abstract

Sequences at both the 5' and 3' ends of mouse histone genes contribute to the expression of individual genes. The 3' sequences required for high expression of the mouse H2a-614 gene are the same as the sequences required for 3'-end formation. When these sequences were substituted for the 3' end of the poorly expressed H2a-291 gene, expression of the H2a-291 gene was increased fivefold. A 65-nucleotide fragment containing the H2a-614 3' processing signal increased expression of the H2a-291 gene when it was placed in the proper orientation downstream of the H2a-291 3' end. The only mRNAs that accumulated from this gene ended at the H2a-291 3' end, which suggests that the transcript is sequentially processed. In an in vitro processing system, the different histone 3' ends showed different processing efficiencies, which correlated with their expression in cells. These results suggest that the efficiency of processing is important in determining the steady-state levels of individual mouse histone mRNAs.The steady-state concentrations of an mRNA are determined by three factors: (i) rate of transcription, (ii) efficiency of processing and transport to the cytoplasm, and (iii) half-life in the cytoplasm. There are numerous examples of the importance of the rate of transcription and half-life to the steady-state concentrations of an mRNA. Much less is known about the efficiency of RNA processing and transport of RNA to the cytoplasm.The replication-dependent histone genes are unique among eucaryotic genes in that they lack intervening sequences and code for mRNAs that are not polyadenylated. Instead, histone mRNAs end in a stem-loop structure, consisting of a 6-base stem and 4-base loop, which has been highly conserved in evolution (4, 15). The 3' end of histone mRNA is formed in a processing event that requires a U7 small nuclear ribonucleoprotein (snRNP) and at least one other component (10,19,22,23). The levels of histone mRNA are regulated during the cell cycle by multiple mechanisms, including alterations in transcription rate, efficiency of 3'-end formation, and changes in mRNA stability (reviewed in references 21 and 26).In mice, the histone genes are present in two clusters on chromosomes 3 and 13 (20). The individual genes on chromosome 3 account for a much higher proportion of the histone mRNA than do the histone genes on chromosome 13 (13), although both sets of genes are regulated coordinately with DNA synthesis during the cell cycle (8, 13). We have recently shown that sequences both 5' and 3' to the genes on chromosome 3 contribute independently to the high level of expression (17). Here we show that the effect of the 3' sequences is to confer a higher efficiency of processing on the histone transcripts from chromosome 3, suggesting that a substantial portion of the transcripts from the genes on chromosome 13 must be degraded in the nucleus and never reach the cytoplasm. and the histone gene constructs, using calcium phosphate precipitation as described previously (1,6). Chinese hamster ovary (CHO) cells we...

Point mutations in the stem-loop at the 3' end of mouse histone mRNA reduce expression by reducing the efficiency of 3' end formation.

“…Fig. 1A, showing the altered nucleotides before the stem-loop and between the stem-loop and the U7 snRNP binding site, regions which are highly variable among different murine histone genes (15). The SLwt mut gene is expressed identically to the wild-type gene (see Fig.…”

“…The stem-loop is usually preceded by at least two A residues and is followed by an AC. The U7 binding site is located 10 to 15 nt 3' of the stem-loop, and both the distance and sequence of the U7 snRNP binding site vary among different murine histone genes (15). The core of the U7 binding site is a purine-rich AAAGAG sequence which base pairs with U7 snRNA (2).…”

“…It is important in regulating the half-life of histone mRNA (9,24) as well as in stimulating the export of histone mRNA from the nucleus (5,32). While the stem-loop sequence is highly conserved among different histone genes, the U7 snRNP binding site varies widely among different histone genes (15). The differences in the U7 binding site result in different efficiencies of pre-mRNA processing, and this contributes to the relative expression of individual histone genes encoding the same histone protein (15).…”

“…The genes containing two processing signals were constructed by placing either the mouse ,B-globin polyadenylation signal or the wildtype histone processing signal 60 nucleotides (nt) 3' of the end of SL(A' 3) mutant processing signal. The cassettes containing the wild-type histone H2a-614 3' end signal (23) or the mouse ,-globin polyadenylation signal have been described previously (15).…”

Knockdown of SLBP results in nuclear retention of histone mRNA

“…A striking aspect of this result is that these mRNAs accumulate to substantial levels. When mutations are introduced into the histone stem-loop that alter SLBP binding (Pandey et al 1994) or the HDE is deleted preventing U7 snRNP binding (Chodchoy et al 1991), or when the HDE is naturally suboptimal (Liu et al 1989), then the unprocessed mRNAs do not accumulate, but rather, must be rapidly degraded. The same is true of improperly processed mRNAs from polyadenylated transcripts.…”