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The histones which pack new DNA during the S phase of animal cells are made from mRNAs that are cleaved at their 3' end but not polyadenylated. Some of the factors used in this reaction are unique to it while others are shared with the polyadenylation process that generates all other mRNAs. Recent work has begun to shed light on how the cell manages the assignment of these common components to the two 3' processing systems, and how it achieves their cell cycle-regulation and recruitment to the histone pre-mRNA. Moreover, recent and older findings reveal multiple connections between the nuclear organization of histone genes, their transcription and 3' end processing as well as the control of cell proliferation., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A set of seven Sm proteins assemble on the Sm-binding site of spliceosomal U snRNAs to form the ring-shaped Sm core. The U7 snRNP involved in histone RNA 3' processing contains a structurally similar but biochemically unique Sm core in which two of these proteins, Sm D1 and D2, are replaced by Lsm10 and by another as yet unknown component. Here we characterize this factor, termed Lsm11, as a novel Sm-like protein with apparently two distinct functions. In vitro studies suggest that its long N-terminal part mediates an important step in histone mRNA 3'-end cleavage, most likely by recruiting a zinc finger protein previously identified as a processing factor. In contrast, the C-terminal part, which comprises two Sm motifs interrupted by an unusually long spacer, is sufficient to assemble with U7, but not U1, snRNA. Assembly of this U7-specific Sm core depends on the noncanonical Sm-binding site of U7 snRNA. Moreover, it is facilitated by a specialized SMN complex that contains Lsm10 and Lsm11 but lacks Sm D1/D2. Thus, the U7-specific Lsm11 protein not only specifies the assembly of the U7 Sm core but also fulfills an important role in U7 snRNP-mediated histone mRNA processing.

Histone pre-mRNA 3' processing is controlled by a hairpin element preceding the processing site that interacts with a hairpin-binding protein (HBP) and a downstream spacer element that serves as anchoring site for the U7 snRNP. In addition, the nucleotides following the hairpin and surrounding the processing site (ACCCA'CA) are conserved among vertebrate histone genes. Single to triple nucleotide mutations of this sequence were tested for their ability to be processed in nuclear extract from animal cells. Changing the first four nucleotides had no qualitative and little if any quantitative effects on histone RNA 3' processing in mouse K21 cell extract, where processing of this gene is virtually independent of the HBP. A gel mobility shift assay revealing HBP interactions and a processing assay in HeLa cell extract (where the contribution of HBP to efficient processing is more important) showed that only one of these mutations, predicted to extend the hairpin by one base pair, affected the interaction with HBP. Mutations in the next three nucleotides affected both the cleavage efficiency and the choice of processing sites. Analysis of these novel sites indicated a preference for the nucleotide 5' of the cleavage site in the order A > C > U > G. Moreover, a guanosine in the 3' position inhibited cleavage. The preference for an A is shared with the cleavage/polyadenylation reaction, but the preference order for the other nucleotides is different [Chen F, MacDonald CC, Wilusz J, 1995, Nucleic Acids Res 23:2614-2620].

We have studied the requirements for efficient histone-specific RNA 3' processing in nuclear extract from mammalian tissue culture cells. Processing is strongly impaired by mutations in the pre-mRNA spacer element that reduce the base-pairing potential with U7 RNA. Moreover, by exchanging the hairpin and spacer elements of two differently processed H4 genes, we find that this difference is exclusively due to the spacer element. Finally, processing is inhibited by the addition of competitor RNAs, if these contain a wild-type spacer sequence, but not if their spacer element is mutated. Conversely, the importance of the hairpin for histone RNA 3' processing is highly variable: A hairpin mutant of the H4-12 gene is processed with almost wild-type efficiency in extract from K21 mouse mastocytoma cells but is strongly affected in HeLa cell extract, whereas an identical hairpin mutant of the H4-1 gene is affected in both extracts. The hairpin defect of H4-12-specific RNA in HeLa cells can be overcome by a compensatory mutation that increases the base complementarity to U7 snRNA. Very similar results were also obtained in RNA competition experiments: processing of H4-12-specific RNA can be competed by RNA carrying a wild-type hairpin element in extract from HeLa, but not K21 cells, whereas processing of H4-1-specific RNA can be competed in both extracts. With two additional histone genes we obtained results that were in one case intermediate and in the other similar to those obtained with H4-1. These results suggest that hairpin binding factor(s) can cooperatively support the ability of U7 snRNPs to form an active processing complex, but is(are) not directly involved in the processing mechanism.

The levels of histone mRNA increase 35-fold as selectively detached mitotic CHO cells progress from mitosis through G1 and into S phase. Using an exogenous gene with a histone 3' end which is not sensitive to transcriptional or half-life regulation, we show that 3' processing is regulated as cells progress from G1 to S phase. The half-life of histone mRNA is similar in G1- and S-phase cells, as measured after inhibition of transcription by actinomycin D (dactinomycin) or indirectly after stabilization by the protein synthesis inhibitor cycloheximide. Taken together, these results suggest that the change in histone mRNA levels between G1- and S-phase cells must be due to an increase in the rate of biosynthesis, a combination of changes in transcription rate and processing efficiency. In G2 phase, there is a rapid 35-fold decrease in the histone mRNA concentration which our results suggest is due primarily to an altered stability of histone mRNA. These results are consistent with a model for cell cycle regulation of histone mRNA levels in which the effects on both RNA 3' processing and transcription, rather than alterations in mRNA stability, are the major mechanisms by which low histone mRNA levels are maintained during G1.

A short histone-like fusion RNA, generated when the RNA 3' processing signal from a mouse histone H4 gene is inserted into a heterologous transcription unit, becomes correctly down-regulated in G1-arrested cells of a temperature-sensitive mouse mastocytoma cell cycle mutant (21-Tb; Stauber et al., EMBO J. 5, 3297-3303 [1986]), due to a specific deficiency in histone RNA processing (Lüscher and Schümperli, EMBO J. 6, 1721-1726 [1987]). In contrast, inhibitors of DNA synthesis, known to stimulate histone mRNA degradation, have little or no effect on the fusion RNA. This RNA can therefore be used to discriminate between regulation by RNA 3' processing and RNA stability, respectively. The fusion RNA is also faithfully regulated in 21-Tb cells arrested in G1 phase by the drug indomethacin or in C127 mouse fibroblasts during a serum starvation experiment. Moreover, nuclear extracts from serum-starved C127 cells show a specific deficiency in a heat-labile component of the histone RNA processing apparatus, similar to that previously observed for temperature-arrested 21-Tb cells. These results suggest that RNA 3' processing is a major determinant for the response of histone mRNA levels to changes in cell proliferation.

In vitro-synthesized transcripts of the sea urchin histone H2A gene with 3' extensions are efficiently and rapidly processed to H2A mRNA with faithful 3' ends in Xenopus laevis oocyte nuclei. Processing requires the presence of a histone-specific dyad symmetry element and of H2A-proximal spacer sequences in the precursor RNA. In DNA injection experiments with a processing-deficient H2A mutant, the transcription products appear to terminate heterogeneously in the first 100-200 base pairs of the post-H2A spacer. Processing of synthetic H3 RNA precursors requires the prior injection of a 60-nucleotide RNA from sea urchin embryos that seems to be a component of a small nuclear ribonucleoprotein.

Post-transcriptional regulation of histone gene expression in a mouse mastocytoma cell cycle mutant (21-Tb) depends largely on conserved DNA sequences that are essential for RNA 3' processing. We have analyzed whether this regulation occurs at the level of RNA 3' processing. We show, by RNase mapping, that nuclear H4 mRNA precursors, which are hardly detectable in total RNA from exponentially dividing cells, accumulate in G1-arrested cells, i.e. when mature mRNAs are drastically reduced. Furthermore, we show that a heat-labile component of the processing apparatus, recently identified in HeLa cell nuclear extracts, is limiting in extracts from G1-arrested 21-Tb cells. In contrast, this activity is in excess in extracts from exponentially dividing cells, whereas both extracts contain similar amounts of snRNPs of the Sm serotype. These fluctuations in the heat-labile activity may generally contribute to proliferation or cell cycle dependent histone gene regulation.

Oligonucleotides derived from the spacer element of the histone RNA 3' processing signal were used to characterize mouse U7 small nuclear RNA (snRNA), i.e., the snRNA component active in 3' processing of histone pre-mRNA. Under RNase H conditions, such oligonucleotides inhibited the processing reaction, indicating the formation of a DNA-RNA hybrid with a functional ribonucleoprotein component. Moreover, these oligonucleotides hybridized to a single nuclear RNA species of approximately 65 nucleotides. The sequence of this RNA was determined by primer extension experiments and was found to bear several structural similarities with sea urchin U7 snRNA. The comparison of mouse and sea urchin U7 snRNA structures yields some further insight into the mechanism of histone RNA 3' processing.