Early evolution of histone mRNA 3' end processing

The replication-dependent histone mRNAs in metazoa are not polyadenylated, in contrast to the bulk of mRNA. Instead, they contain an RNA stem-loop (SL) structure close to the 3' end of the mature RNA, and this 3' end is generated by cleavage using a machinery involving the U7 snRNP and protein factors such as the stem-loop binding protein (SLBP). This machinery of 3' end processing is related to that of polyadenylation as protein components are shared between the systems. It is commonly believed that histone 3' end processing is restricted to metazoa and green algae. In contrast, polyadenylation is ubiquitous in Eukarya. However, using computational approaches, we have now identified components of histone 3' end processing in a number of protozoa. Thus, the histone mRNA stem-loop structure as well as the SLBP protein are present in many different protozoa, including Dictyostelium, alveolates, Trypanosoma, and Trichomonas. These results show that the histone 3' end processing machinery is more ancient than previously anticipated and can be traced to the root of the eukaryotic phylogenetic tree. We also identified histone mRNAs from both metazoa and protozoa that are polyadenylated but also contain the signals characteristic of histone 3' end processing. These results provide further evidence that some histone genes are regulated at the level of 3' end processing to produce either polyadenylated RNAs or RNAs with the 3' end characteristic of replication-dependent histone mRNAs.     

Differences and similarities between Drosophila and mammalian 3' end processing of histone pre-mRNAs

We used nuclear extracts from Drosophila Kc cells to characterize 3' end processing of Drosophila histone pre-mRNAs. Drosophila SLBP plays a critical role in recruiting the U 7 snRNP to the pre-mRNA and is essential for processing all five Drosophila histone pre-mRNAs. The Drosophila processing machinery strongly prefers cleavage after a fourth nucleotide following the stem-loop and favors an adenosine over pyrimidines in this position. Increasing the distance between the stem-loop and the HDE does not result in a corresponding shift of the cleavage site, suggesting that in Drosophila processing the U 7 snRNP does not function as a molecular ruler. Instead, SLBP directs the cleavage site close to the stem-loop. The upstream cleavage product generated in Drosophila nuclear extracts contains a 3' OH, and the downstream cleavage product is degraded by a nuclease dependent on the U 7 snRNP, suggesting that the cleavage factor has been conserved between Drosophila and mammalian processing. A 2'O-methyl oligonucleotide complementary to the first 17 nt of the Drosophila U 7 snRNA was not able to deplete the U 7 snRNP from Drosophila nuclear extracts, suggesting that the 5' end of the Drosophila U 7 snRNA is inaccessible. This oligonucleotide selectively inhibited processing of only two Drosophila pre-mRNAs and had no effect on processing of the other three pre-mRNAs. Together, these studies demonstrate that although Drosophila and mammalian histone pre-mRNA processing share common features, there are also significant differences, likely reflecting divergence in the mechanism of 3' end processing between vertebrates and invertebrates.     

Formation of the 3' end of histone mRNA

All metazoan messenger RNAs, with the exception of the replication-dependent histone mRNAs, terminate at the 3' end with a poly(A) tail. Replication-dependent histone mRNAs end instead in a conserved 26-nucleotide sequence that contains a 16-nucleotide stem-loop. Formation of the 3' end of histone mRNA occurs by endonucleolytic cleavage of pre-mRNA releasing the mature mRNA from the chromatin template. Cleavage requires several trans-acting factors, including a protein, the stem-loop binding protein (SLBP), which binds the 26-nucleotide sequence; and a small nuclear RNP, U7 snRNP. There are probably additional factors also required for cleavage. One of the functions of the SLBP is to stabilize binding of the U7 snRNP to the histone pre-mRNA. In the nucleus, both U7 snRNP and SLBP are present in coiled bodies, structures that are associated with histone genes and may play a direct role in histone pre-mRNA processing in vivo. One of the major regulatory events in the cell cycle is regulation of histone pre-mRNA processing, which is at least partially mediated by cell-cycle regulation of the levels of the SLBP protein.     

3' end processing of Drosophila melanogaster histone pre-mRNAs: requirement for phosphorylated Drosophila stem-loop binding protein and coevolution of the histone pre-mRNA processing system

Synthetic pre-mRNAs containing the processing signals encoded by Drosophila melanogaster histone genes undergo efficient and faithful endonucleolytic cleavage in nuclear extracts prepared from Drosophila cultured cells and 0- to 13-h-old embryos. Biochemical requirements for the in vitro cleavage are similar to those previously described for the 3' end processing of mammalian histone pre-mRNAs. Drosophila 3' end processing does not require ATP and occurs in the presence of EDTA. However, in contrast to mammalian processing, Drosophila processing generates the final product ending four nucleotides after the stem-loop. Cleavage of the Drosophila substrates is abolished by depleting the extract of the Drosophila stem-loop binding protein (dSLBP), indicating that both dSLBP and the stem-loop structure in histone pre-mRNA are essential components of the processing machinery. Recombinant dSLBP expressed in insect cells by using the baculovirus system efficiently complements the depleted extract. Only the RNA-binding domain plus the 17 amino acids at the C terminus of dSLBP are required for processing. The full-length dSLBP expressed in insect cells is quantitatively phosphorylated on four residues in the C-terminal region. Dephosphorylation of the recombinant dSLBP reduces processing activity. Human and Drosophila SLBPs are not interchangeable and strongly inhibit processing in the heterologous extracts. The RNA-binding domain of the dSLBP does not substitute for the RNA-binding domain of the human SLBP in histone pre-mRNA processing in mammalian extracts. In addition to the stem-loop structure and dSLBP, 3' processing in Drosophila nuclear extracts depends on the presence of a short stretch of purines located ca. 20 nucleotides downstream from the stem, and an Sm-reactive factor, most likely the Drosophila counterpart of vertebrate U7 snRNP.     

The polyribosomal protein bound to the 3' end of histone mRNA can function in histone pre-mRNA processing.

Cell cycle-regulated histone mRNAs end in a conserved 26-nt sequence that can form a stem-loop with a six-base stem and a four-base loop. The 3' end of histone mRNA has distinct functions in the nucleus and in the cytoplasm. In the nucleus it functions in pre-mRNA processing and transport, whereas in the cytoplasm it functions in translation and regulation of histone mRNA stability. The stem-loop binding protein (SLBP), present in both nuclei and polyribosomes, is likely the trans-acting factor that binds to the 3' end of mature histone mRNA and mediates its function. A nuclear extract that efficiently processes histone pre-mRNA was prepared from mouse myeloma cells. The factor(s) that bind to the 3' end of histone mRNA can be depleted from this extract using a biotinylated oligonucleotide containing the conserved stem-loop sequence. Using this depleted extract which is deficient in histone pre-mRNA processing, we show that SLBP found in polyribosomes can restore processing, suggesting that SLBP associates with histone pre-mRNA in the nucleus, participates in processing, and then accompanies the mature mRNA to the cytoplasm.     

A 3' exonuclease that specifically interacts with the 3' end of histone mRNA

Metazoan histone mRNAs end in a highly conserved stem-loop structure followed by ACCCA. Previous studies have suggested that the stem-loop binding protein (SLBP) is the only protein binding this region. Using RNA affinity purification, we identified a second protein, designated 3'hExo, that contains a SAP and a 3' exonuclease domain and binds the same sequence. Strikingly, 3'hExo can bind the stem-loop region both separately and simultaneously with SLBP. Binding of 3'hExo requires the terminal ACCCA, whereas binding of SLBP requires the 5' side of the stem-loop region. Recombinant 3'hExo degrades RNA substrates in a 3'-5' direction and has the highest activity toward the wild-type histone mRNA. Binding of SLBP to the stem-loop at the 3' end of RNA prevents its degradation by 3'hExo. These features make 3'hExo a primary candidate for the exonuclease that initiates rapid decay of histone mRNA upon completion and/or inhibition of DNA replication.     

Formation of the 3' end of histone mRNA: getting closer to the end

Nearly all eukaryotic mRNAs end with a poly(A) tail that is added to their 3' end by the ubiquitous cleavage/polyadenylation machinery. The only known exceptions to this rule are metazoan replication-dependent histone mRNAs, which end with a highly conserved stem-loop structure. This distinct 3' end is generated by specialized 3' end processing machinery that cleaves histone pre-mRNAs 4-5 nucleotides downstream of the stem-loop and consists of the U7 small nuclear RNP (snRNP) and number of protein factors. Recently, the U7 snRNP has been shown to contain a unique Sm core that differs from that of the spliceosomal snRNPs, and an essential heat labile processing factor has been identified as symplekin. In addition, cross-linking studies have pinpointed CPSF-73 as the endonuclease, which catalyzes the cleavage reaction. Thus, many of the critical components of the 3' end processing machinery are now identified. Strikingly, this machinery is not as unique as initially thought but contains at least two factors involved in cleavage/polyadenylation, suggesting that the two mechanisms have a common evolutionary origin. The greatest challenge that lies ahead is to determine how all these factors interact with each other to form a catalytically competent processing complex capable of cleaving histone pre-mRNAs.     

Regulation of yeast mRNA 3' end processing by phosphorylation

Recent studies have found that the phosphatase Glc7 associates with the yeast cleavage/polyadenylation factor (CPF), but the role of Glc7 in 3' end processing has not been investigated. Here, we report that depletion of Glc7 causes shortened poly(A) tails in vivo and accumulation of phosphorylated Pta1, a CPF subunit. Removal of Glc7 also gives extract defective for poly(A) addition but normal for cleavage at the poly(A) site. Polyadenylation is rescued by addition of Glc7 or Pta1, but not by phosphorylated Pta1. Moreover, Ypi1, a Glc7-specific inhibitor, or the Cka1 kinase blocks poly(A) addition in wild-type (wt) extract. Pta1 interacts physically and genetically with Glc7, suggesting that Pta1 may also regulate Glc7 or recruit it to CPF. A weakened association of Fip1 with phosphorylated CPF may explain the specific effect on polyadenylation. These results support a model in which poly(A) synthesis is controlled by cycles of phosphorylation and dephosphorylation that require the action of Glc7.     

Characterization of 3'hExo, a 3' exonuclease specifically interacting with the 3' end of histone mRNA

The 3' end of mammalian histone mRNAs consisting of a conserved stem-loop and a terminal ACCCA interacts with a recently identified human 3' exonuclease designated 3'hExo. The sequence-specific interaction suggests that 3'hExo may participate in the degradation of histone mRNAs. ERI-1, a Caenorhabditis elegans homologue of 3'hExo, has been implicated in degradation of small interfering RNAs. We introduced a number of mutations to 3'hExo to identify residues required for RNA binding and catalysis. To assure that the introduced mutations specifically target one of these two activities of 3'hExo rather than cause global structural defects, the mutant proteins were tested in parallel for the ability both to bind the stem-loop RNA and to degrade RNA substrates. Our analysis confirms that 3'hExo is a member of the DEDDh family of 3' exonucleases. Specific binding to the RNA requires the SAP domain and two lysines located immediately to its C terminus. 3'hExo binds with the highest affinity to the wild-type 3' end of histone mRNA, and any changes to this sequence reduce efficiency of binding. 3'hExo has only residual, if any, 3' exonuclease activity on DNA substrates and localizes mostly to the cytoplasm, suggesting that in vivo it performs exclusively RNA-specific functions. Efficient degradation of RNA substrates by 3'hExo requires 2' and 3' hydroxyl groups at the last nucleotide. 3'hExo removes 3' overhangs of small interfering RNAs, whereas the double-stranded region is resistant to the enzymatic activity.     

Nucleophosmin deposition during mRNA 3' end processing influences poly(A) tail length

During polyadenylation, the multi-functional protein nucleophosmin (NPM1) is deposited onto all cellular mRNAs analysed to date. Premature termination of poly(A) tail synthesis in the presence of cordycepin abrogates deposition of the protein onto the mRNA, indicating natural termination of poly(A) addition is required for NPM1 binding. NPM1 appears to be a bona fide member of the complex involved in 3' end processing as it is associated with the AAUAAA-binding CPSF factor and can be co-immunoprecipitated with other polyadenylation factors. Furthermore, reduction in the levels of NPM1 results in hyperadenylation of mRNAs, consistent with alterations in poly(A) tail chain termination. Finally, knockdown of NPM1 results in retention of poly(A)(+) RNAs in the cell nucleus, indicating that NPM1 influences mRNA export. Collectively, these data suggest that NPM1 has an important role in poly(A) tail length determination and may help network 3' end processing with other aspects of nuclear mRNA maturation.