The Prolyl Isomerase Pin1 Targets Stem-Loop Binding Protein (SLBP) To Dissociate the SLBP-Histone mRNA Complex Linking Histone mRNA Decay with SLBP Ubiquitination

Histone mRNAs are rapidly degraded at the end of S phase, and a 26-nucleotide stem-loop in the 3′ untranslated region is a key determinant of histone mRNA stability. This sequence is the binding site for stem-loop binding protein (SLBP), which helps to recruit components of the RNA degradation machinery to the histone mRNA 3′ end. SLBP is the only protein whose expression is cell cycle regulated during S phase and whose degradation is temporally correlated with histone mRNA degradation. Here we report that chemical inhibition of the prolyl isomerase Pin1 or downregulation of Pin1 by small interfering RNA (siRNA) increases the mRNA stability of all five core histone mRNAs and the stability of SLBP. Pin1 regulates SLBP polyubiquitination via the Ser20/Ser23 phosphodegron in the N terminus. siRNA knockdown of Pin1 results in accumulation of SLBP in the nucleus. We show that Pin1 can act along with protein phosphatase 2A (PP2A) in vitro to dephosphorylate a phosphothreonine in a conserved TPNK sequence in the SLBP RNA binding domain, thereby dissociating SLBP from the histone mRNA hairpin. Our data suggest that Pin1 and PP2A act to coordinate the degradation of SLBP by the ubiquitin proteasome system and the exosome-mediated degradation of the histone mRNA by regulating complex dissociation.     

SLIP1, a factor required for activation of histone mRNA translation by the stem-loop binding protein

Replication-dependent histone mRNAs are the only eukaryotic cellular mRNAs that are not polyadenylated, ending instead in a conserved stem-loop. The 3′ end of histone mRNA is required for histone mRNA translation, as is the stem-loop binding protein (SLBP), which binds the 3′ end of histone mRNA. We have identified five conserved residues in a 15-amino-acid region in the amino-terminal portion of SLBP, each of which is required for translation. Using a yeast two-hybrid screen, we identified a novel protein, SLBP-interacting protein 1 (SLIP1), that specifically interacts with this region. Mutations in any of the residues required for translation reduces SLIP1 binding to SLBP. The expression of SLIP1 in Xenopus oocytes together with human SLBP stimulates translation of a reporter mRNA ending in the stem-loop but not a reporter with a poly(A) tail. The expression of SLIP1 in HeLa cells also stimulates the expression of a green fluorescent protein reporter mRNA ending in a stem-loop. RNA interference-mediated downregulation of endogenous SLIP1 reduces the rate of translation of endogenous histone mRNA and also reduces cell viability. SLIP1 may function by bridging the 3′ end of the histone mRNA with the 5′ end of the mRNA, similar to the mechanism of translation of polyadenylated mRNAs.     

Two Xenopus Proteins That Bind the 3′ End of Histone mRNA: Implications for Translational Control of Histone Synthesis during Oogenesis

Translationally inactive histone mRNA is stored in frog oocytes, and translation is activated at oocyte maturation. The replication-dependent histone mRNAs are not polyadenylated and end in a conserved stem-loop structure. There are two proteins (SLBPs) which bind the 3′ end of histone mRNA in frog oocytes. SLBP1 participates in pre-mRNA processing in the nucleus. SLBP2 is oocyte specific, is present in the cytoplasm, and does not support pre-mRNA processing in vivo or in vitro. The stored histone mRNA is bound to SLBP2. As oocytes mature, SLBP2 is degraded and a larger fraction of the histone mRNA is bound to SLBP1. The mechanism of activation of translation of histone mRNAs may involve exchange of SLBPs associated with the 3′ end of histone mRNA.     

The mRNP remodeling mediated by UPF1 promotes rapid degradation of replication-dependent histone mRNA

Histone biogenesis is tightly controlled at multiple steps to maintain the balance between the amounts of DNA and histone protein during the cell cycle. In particular, translation and degradation of replication-dependent histone mRNAs are coordinately regulated. However, the underlying molecular mechanisms remain elusive. Here, we investigate remodeling of stem-loop binding protein (SLBP)-containing histone mRNPs occurring during the switch from the actively translating mode to the degradation mode. The interaction between a CBP80/20-dependent translation initiation factor (CTIF) and SLBP, which is important for efficient histone mRNA translation, is disrupted upon the inhibition of DNA replication or at the end of S phase. This disruption is mediated by competition between CTIF and UPF1 for SLBP binding. Further characterizations reveal hyperphosphorylation of UPF1 by activated ATR and DNA-dependent protein kinase upon the inhibition of DNA replication interacts with SLBP more strongly, promoting the release of CTIF and eIF3 from SLBP-containing histone mRNP. In addition, hyperphosphorylated UPF1 recruits PNRC2 and SMG5, triggering decapping followed by 5′-to-3′ degradation of histone mRNAs. The collective observations suggest that both inhibition of translation and recruitment of mRNA degradation machinery during histone mRNA degradation are tightly coupled and coordinately regulated by UPF1 phosphorylation.     

Loss of the Histone Pre-mRNA Processing Factor Stem-Loop Binding Protein in Drosophila Causes Genomic Instability and Impaired Cellular Proliferation

Background

Metazoan replication-dependent histone mRNAs terminate in a conserved stem-loop structure rather than a polyA tail. Formation of this unique mRNA 3′ end requires Stem-loop Binding Protein (SLBP), which directly binds histone pre-mRNA and stimulates 3′ end processing. The 3′ end stem-loop is necessary for all aspects of histone mRNA metabolism, including replication coupling, but its importance to organism fitness and genome maintenance in vivo have not been characterized.

Methodology/Principal Findings

In Drosophila, disruption of the Slbp gene prevents normal histone pre-mRNA processing and causes histone pre-mRNAs to utilize the canonical 3′ end processing pathway, resulting in polyadenylated histone mRNAs that are no longer properly regulated. Here we show that Slbp mutants display genomic instability, including loss of heterozygosity (LOH), increased presence of chromosome breaks, tetraploidy, and changes in position effect variegation (PEV). During imaginal disc growth, Slbp mutant cells show defects in S phase and proliferate more slowly than control cells.

Conclusions/Significance

These data are consistent with a model in which changing the 3′ end of histone mRNA disrupts normal replication-coupled histone mRNA biosynthesis and alters chromatin assembly, resulting in genomic instability, inhibition of cell proliferation, and impaired development.     

The C-terminal extension of Lsm4 interacts directly with the 3′ end of the histone mRNP and is required for efficient histone mRNA degradation

Metazoan replication-dependent histone mRNAs are the only known eukaryotic mRNAs that lack a poly(A) tail, ending instead in a conserved stem–loop sequence, which is bound to the stem–loop binding protein (SLBP) on the histone mRNP. Histone mRNAs are rapidly degraded when DNA synthesis is inhibited in S phase in mammalian cells. Rapid degradation of histone mRNAs is initiated by oligouridylation of the 3′ end of histone mRNAs and requires the cytoplasmic Lsm1-7 complex, which can bind to the oligo(U) tail. An exonuclease, 3′hExo, forms a ternary complex with SLBP and the stem–loop and is required for the initiation of histone mRNA degradation. The Lsm1-7 complex is also involved in degradation of polyadenylated mRNAs. It binds to the oligo(A) tail remaining after deadenylation, inhibiting translation and recruiting the enzymes required for decapping. Whether the Lsm1-7 complex interacts directly with other components of the mRNP is not known. We report here that the C-terminal extension of Lsm4 interacts directly with the histone mRNP, contacting both SLBP and 3′hExo. Mutants in the C-terminal tail of Lsm4 that prevent SLBP and 3′hExo binding reduce the rate of histone mRNA degradation when DNA synthesis is inhibited.     

Changes in the stem-loop at the 3' terminus of histone mRNA affects its nucleocytoplasmic transport and cytoplasmic regulation.

The stem-loop structure at the 3' end of replication-dependent histone mRNA is required for efficient pre-mRNA processing, localization of histone mRNA to the polyribosomes, and regulation of histone mRNA degradation. A protein, the stem-loop binding protein (SLBP), binds the 3' end of histone mRNA and is thought to mediate some or all of these processes. A mutant histone mRNA with two nucleotide changes in the loop was constructed and found to be transported inefficiently to the cytoplasm. The mutant histone mRNA, unlike the wild-type histone mRNA, was not rapidly degraded when DNA synthesis is inhibited, and was not stabilized upon inhibition of protein synthesis. The stem-loop binding protein (SLBP) has between a 20-50 fold greater affinity for the wild type histone stem-loop structure than for the mutant stem-loop structure, suggesting that the alteration in the efficiency of transport and the normal degradation pathway in histone mRNA may be due to the reduced affinity of the mutant stem-loop for the SLBP.     

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.     

3′-End processing of histone pre-mRNAs in Drosophila: U7 snRNP is associated with FLASH and polyadenylation factors

3′-End cleavage of animal replication-dependent histone pre-mRNAs is controlled by the U7 snRNP. Lsm11, the largest component of the U7-specific Sm ring, interacts with FLASH, and in mammalian nuclear extracts these two proteins form a platform that recruits the CPSF73 endonuclease and other polyadenylation factors to the U7 snRNP. FLASH is limiting, and the majority of the U7 snRNP in mammalian extracts exists as a core particle consisting of the U7 snRNA and the Sm ring. Here, we purified the U7 snRNP from Drosophila nuclear extracts and characterized its composition by mass spectrometry. In contrast to the mammalian U7 snRNP, a significant fraction of the Drosophila U7 snRNP contains endogenous FLASH and at least six subunits of the polyadenylation machinery: symplekin, CPSF73, CPSF100, CPSF160, WDR33, and CstF64. The same composite U7 snRNP is recruited to histone pre-mRNA for 3′-end processing. We identified a motif in Drosophila FLASH that is essential for the recruitment of the polyadenylation complex to the U7 snRNP and analyzed the role of other factors, including SLBP and Ars2, in 3′-end processing of Drosophila histone pre-mRNAs. SLBP that binds the upstream stem–loop structure likely recruits a yet-unidentified essential component(s) to the processing machinery. In contrast, Ars2, a protein previously shown to interact with FLASH in mammalian cells, is dispensable for processing in Drosophila. Our studies also demonstrate that Drosophila symplekin and three factors involved in cleavage and polyadenylation—CPSF, CstF, and CF I_m—are present in Drosophila nuclear extracts in a stable supercomplex.     

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.     

A novel function for the U2AF 65 splicing factor in promoting pre-mRNA 3'-end processing

Splicing and 3′-end processing (including cleavage and polyadenylation) of vertebrate pre-mRNAs are tightly coupled events that contribute to the extensive molecular network that coordinates gene expression. Sequences within the terminal intron of genes are essential to stimulate pre-mRNA 3′-end processing, although the factors mediating this effect are unknown. Here, we show that the pyrimidine tract of the last splice acceptor site of the human β-globin gene is necessary to stimulate mRNA 3′-end formation in vivo and binds the U2AF 65 splicing factor. Naturally occurring β-thalassaemia-causing mutations within the pyrimidine tract reduces both U2AF 65 binding and 3′-end cleavage efficiency. Significantly, a fusion protein containing U2AF 65, when tethered upstream of a cleavage/polyadenylation site, increases 3′-end cleavage efficiency in vitro and in vivo. Therefore, we propose that U2AF 65 promotes 3′-end processing, which contributes to 3′-terminal exon definition.