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

Abstract

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.The replication-dependent histone genes differ from other histone genes in that they lack intervening sequences and they encode mRNAs which are not polyadenylated (18). Instead, the histone mRNAs end in a highly conserved stem-loop structure, a six-base stem with a four-base loop (1). The 3' end of mammalian histone mRNA is formed by an endonucleolytic cleavage which requires the stem-loop and a 3' purine-rich region which interacts with U7 small nuclear RNA (snRNA) (2,4,6,22). There are at least three components required for processing, the stem-loop-binding protein (SLBP), the U7 small nuclear ribonucleoprotein (snRNP), and a heat-labile factor (7,16,19,33).The stem-loop at the 3' end of histone mRNA provides some of the functions of the poly(A) tail found in other mRNAs (13,17). 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 efficiency of histone pre-mRNA processing is also regulated during the cell cycle. In serum-starved fibroblasts (29) and in continuously cycling CHO cells (11), histone mRNA is poorly processed during G, phase and is then efficiently processed during S phase. The increase in processing efficiency plays a major role in the rapid accumulation of histone mRNA at the start of S phase. Decreased processing efficiency is due to a lack of the heat-labile factor (7, 16) an...

The protein that binds the 3' end of histone mRNA: a novel RNA-binding protein required for histone pre-mRNA processing.

“…The stem-loop at the 3' end of the histone mRNA is necessary for efficient 3' end formation in vivo (Pandey et al 1994) and is necessary and sufficient for regulation of histone mRNA degradation (Pandey and Marzluff 1987). We have detected a specific protein, termed SLBP, that interacts with the 3' end of histone mRNA.…”

Characterization of Two Nuclear Proteins that Interact with Cytochrome P‐450 1A2 mRNA

“…IB), it is tempting to speculate that this putative secondary structure is important for the binding of the proteins. One example of the importance of such a secondary structure is the 3' end of histone RNA, where the presence and integrity of a stem-loop is essential for the post-transcriptional regulation of the mRNA [21,361 and for the binding of a stem-loop-binding protein [37] possibly involved in histone pre-mRNA processing [38].…”

Formation of the 3′ end of histone mRNA: Getting closer to the end

“…On the other hand, substrates that form strong duplexes with the U7 snRNA can be processed in vitro with a significant efficiency in the absence of SLBP Melin et al, 1992;Spycher et al, 1994;Streit et al, 1993). Under in vivo conditions, SLBP is likely to be an essential factor in processing of all histone pre-mRNAs regardless of the HDE sequence and the extent of base pairing to the U7 snRNA (Pandey et al, 1994). Following processing, SLBP remains associated with the stem-loop at the 3' end and accompanies the mature histone mRNA to the cytoplasm (Dominski et al, 1995;Erkmann et al, 2005;Whitfield et al, 2004), where it stimulates its translation into histone proteins (Gorgoni et al, 2005;Sanchez and Marzluff, 2002).…”