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The accumulation and storage of maternal mRNA is crucial for oocyte maturation and embryonic development. PATL2 is an oocyte-specific RNA-binding protein, and previous studies have confirmed that PATL2 mutation in humans and knockout mice cause oocyte maturation arrest or embryonic development arrest, respectively. However, the physiological function of PATL2 in the process of oocyte maturation and embryonic development is largely unknown. Here, we report that PATL2 is highly expressed in growing oocytes and couples with EIF4E and CPEB1 to regulate maternal mRNA expression in immature oocytes. The germinal vesicle oocytes from Patl2-/- mice exhibit decreasing maternal mRNA expression and reduced levels of protein synthesis. We further confirmed that PATL2 phosphorylation occurs in the oocyte maturation process and identified the S279 phosphorylation site using phosphoproteomics. We found that the S279D mutation decreased the protein level of PATL2 and led to subfertility in Palt2S279D knock-in mice. Our work reveals the previously unrecognized role of PATL2 in regulating the maternal transcriptome and shows that phosphorylation of PATL2 leads to the regulation of PATL2 protein levels via ubiquitin-mediated proteasomal degradation in oocytes., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Electronegative clusters (ENCs) made up of acidic residues and/or phosphorylation sites are the most abundant repetitive sequences in RNA-binding proteins. Previous studies have indicated that ENCs inhibit RNA binding for structured RNA-binding domains (RBDs). However, this is not the case for the unstructured RBD in histone pre-mRNA stem-loop binding protein (SLBP). The SLBP RBD contains 70 amino acids and is followed by a phosphorylatable ENC. ENC phosphorylation increases RNA-binding affinity of SLBP to the sub-picomolar range. In this study, we use NMR and molecular dynamics simulations to elucidate the mechanism for this tight binding. Our NMR data demonstrate that the ENC transiently folds apo SLBP into an RNA-bound resembling state. We find that in the RNA-bound state, the phosphorylated ENC interacts with the loop region opposite to the RNA-binding site. This allosteric interaction stabilizes the complex and therefore enhances RNA binding. To evaluate the generality of our findings, we graft an ENC onto endoribonuclease homolog 1's first double-stranded RNA-binding motif (DRBM1), an unstructured RBD that shares no homology with SLBP. We find that the engineered ENC increases the folded species of DRBM1 and inhibits RNA binding. On the contrary, introducing basic residues to DRBM1 makes the domain more unfolded, enhances RNA binding, and mitigates the inhibitory effect of the engineered ENC. In summary, our study suggests that ENCs promote folding of unstructured RNA-binding domains, and their effects on RNA binding depend on the electropositive charges on the RBD surface., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Heterodimerization of RNA binding proteins Nrd1 and Nab3 is essential to communicate the RNA recognition in the nascent transcript with the Nrd1 recognition of the Ser 5 -phosphorylated Rbp1 C-terminal domain in RNA polymerase II. The structure of a Nrd1-Nab3 chimera reveals the basis of heterodimerization, filling a missing gap in knowledge of this system. The free form of the Nrd1 interaction domain of Nab3 (NRID) forms a multi-state three-helix bundle that is clamped in a single conformation upon complex formation with the Nab3 interaction domain of Nrd1 (NAID). The latter domain forms two long helices that wrap around NRID, resulting in an extensive protein-protein interface that would explain the highly favorable free energy of heterodimerization. Mutagenesis of some conserved hydrophobic residues involved in the heterodimerization leads to temperature-sensitive phenotypes, revealing the importance of this interaction in yeast cell fitness. The Nrd1-Nab3 structure resembles the previously reported Rna14/Rna15 heterodimer structure, which is part of the poly(A)-dependent termination pathway, suggesting that both machineries use similar structural solutions despite they share little sequence homology and are potentially evolutionary divergent., (© 2022 Chaves-Arquero et al.)

The induction of ferroptosis is considered a new strategy for cancer treatment. Cytoplasmic polyadenylation element binding protein 1 (CPEB1) is a post-transcriptional regulatory factor, whose low expression has been reported to link to the enhanced metastasis and angiogenesis of gastric cancer (GC). In this study, to explore the role of CPEB1 in ferroptosis, GC cells with overexpressed or silenced CPEB1 expression were treated with erastin, a classic ferroptosis inducer. The results showed that erastin dose-dependently decreased the viability of four GC cell lines (AGS, SNU-1, Hs-746 T, and HGC-27), suggesting that ferroptosis could be triggered in these GC cells. Interestingly, HGC-27 cells overexpressing CPEB1 were more sensitive to erastin, generated more lipid reactive oxygen species (ROS) and malondialdehyde (MDA), and their glutathione peroxidase 4 (Gpx4) expression and GSH content were reduced. Contrarily, CPEB1-silenced AGS cells were more resistant to erastin. Mechanically, we demonstrated that CPEB1 overexpression reduced the expression of twist1, an inhibitor of activating transcription factor 4 (ATF4), thereby activating the ATF4/ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 1 (CHAC1) pathway (CHAC1, a molecule known to induce GSH degradation). Furthermore, re-expression of twist1 in GC cells impaired the effects of CPEB1 overexpression in presence of erastin. Additionally, similar to the in vitro results, the growth-inhibiting effects of erastin on GC xenografted tumors were also augmented by CPEB1 overexpression in vivo. Collectively, we demonstrate that CPEB1 facilitates erastin-induced ferroptosis by inhibiting twist1., (© 2021 International Union of Biochemistry and Molecular Biology.)

Human bocavirus 1 (HBoV1), which belongs to the genus Bocaparvovirus of the Parvoviridae family, causes acute respiratory tract infections in young children. In vitro , HBoV1 infects polarized primary human airway epithelium (HAE) cultured at an air-liquid interface (HAE-ALI). HBoV1 encodes a small nonstructural protein, nuclear protein 1 (NP1), that plays an essential role in the maturation of capsid protein (VP)-encoding mRNAs and viral DNA replication. In this study, we determined the broad interactome of NP1 using the proximity-dependent biotin identification (BioID) assay combined with mass spectrometry (MS). We confirmed that two host mRNA processing factors, DEAH-box helicase 15 (DHX15) and cleavage and polyadenylation specificity factor 6 (CPSF6; also known as CFIm68), a subunit of the cleavage factor Im complex (CFIm), interact with HBoV1 NP1 independently of any DNA or mRNAs. Knockdown of CPSF6 significantly decreased the expression of capsid protein but not that of DHX15. We further demonstrated that NP1 directly interacts with CPSF6 in vitro and colocalizes within the virus replication centers. Importantly, we revealed a novel role of CPSF6 in the nuclear import of NP1, in addition to the critical role of CPSF6 in NP1-facilitated maturation of VP-encoding mRNAs. Thus, our study suggests that CPSF6 interacts with NP1 to escort NP1 imported into the nucleus for its function in the modulation of viral mRNA processing and viral DNA replication. IMPORTANCE Human bocavirus 1 (HBoV1) is one of the significant pathogens causing acute respiratory tract infections in young children worldwide. HBoV1 encodes a small nonstructural protein (NP1) that plays an important role in the maturation of viral mRNAs encoding capsid proteins as well as in viral DNA replication. Here, we identified a critical host factor, CPSF6, that directly interacts with NP1, mediates the nuclear import of NP1, and plays a role in the maturation of capsid protein-encoding mRNAs in the nucleus. The identification of the direct interaction between viral NP1 and host CPSF6 provides new insights into the mechanism by which a viral small nonstructural protein facilitates the multiple regulation of viral gene expression and replication and reveals a novel target for potent antiviral drug development., (Copyright © 2020 American Society for Microbiology.)

Most eukaryotic messenger RNA precursors must undergo 3'-end cleavage and polyadenylation for maturation. We and others recently reported the structure of the AAUAAA polyadenylation signal (PAS) in complex with the protein factors CPSF-30, WDR33, and CPSF-160, revealing the molecular mechanism for this recognition. Here we have characterized in detail the interactions between the PAS RNA and the protein factors using fluorescence polarization experiments. Our studies show that AAUAAA is recognized with ∼3 nM affinity by the CPSF-160-WDR33-CPSF-30 ternary complex. Variations in the RNA sequence can greatly reduce the affinity. Similarly, mutations of CPSF-30 residues that have van der Waals interactions with the bases of AAUAAA also lead to substantial reductions in affinity. Finally, our studies confirm that both CPSF-30 and WDR33 are required for high-affinity binding of the PAS RNA, while these two proteins alone and their binary complexes with CPSF-160 have much lower affinity for the RNA., (© 2019 Hamilton et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

As the female gamete, meiotic oocytes provide not only half of the genome but also almost all stores for fertilization and early embryonic development. Because de novo mRNA transcription is absent in oocyte meiosis, protein-level regulations, especially the ubiquitin proteasome system, are more crucial. As the largest family of ubiquitin E3 ligases, Skp1-Cullin-F-box complexes recognize their substrates via F-box proteins with substrate-selected specificity. However, the variety of F-box proteins and their unknown substrates hinder our understanding of their functions. In this report, we find that Fbxo30, a new member of F-box proteins, is enriched in mouse oocytes, and its expression level declines substantially after the metaphase of the first meiosis (MI). Notably, depletion of Fbxo30 causes significant chromosome compaction accompanied by chromosome segregation failure and arrest at the MI stage, and this arrest is not caused by over-activation of spindle assembly checkpoint. Using immunoprecipitation and mass spectrometric analysis, we identify stem-loop-binding protein (SLBP) as a novel substrate of Fbxo30. SLBP overexpression caused by Fbxo30 depletion results in a remarkable overload of histone H3 on chromosomes that excessively condenses chromosomes and inhibits chromosome segregation. Our finding uncovers an unidentified pathway-controlling chromosome segregation and cell progress.

The cellular response to DNA damage is an intricate mechanism that involves the interplay among several pathways. In this study, we provide evidence of the roles of the polyadenylation factor cleavage stimulation factor 50 (CstF-50) and the ubiquitin (Ub) escort factor p97 as cofactors of BRCA1/BARD1 E3 Ub ligase, facilitating chromatin remodeling during the DNA damage response (DDR). CstF-50 and p97 formed complexes with BRCA1/BARD1, Ub, and some BRCA1/BARD1 substrates, such as RNA polymerase (RNAP) II and histones. Furthermore, CstF-50 and p97 had an additive effect on the activation of the ubiquitination of these BRCA1/BARD1 substrates during DDR. Importantly, as a result of these functional interactions, BRCA1/BARD1/CstF-50/p97 had a specific effect on the chromatin structure of genes that were differentially expressed. This study provides new insights into the roles of RNA processing, BRCA1/BARD1, the Ub pathway, and chromatin structure during DDR., (Copyright © 2018 Fonseca et al.)

3' polyadenylation is a key step in eukaryotic mRNA biogenesis. In mammalian cells, this process is dependent on the recognition of the hexanucleotide AAUAAA motif in the pre-mRNA polyadenylation signal by the cleavage and polyadenylation specificity factor (CPSF) complex. A core CPSF complex comprising CPSF160, WDR33, CPSF30 and Fip1 is sufficient for AAUAAA motif recognition, yet the molecular interactions underpinning its assembly and mechanism of PAS recognition are not understood. Based on cross-linking-coupled mass spectrometry, crystal structure of the CPSF160-WDR33 subcomplex and biochemical assays, we define the molecular architecture of the core human CPSF complex, identifying specific domains involved in inter-subunit interactions. In addition to zinc finger domains in CPSF30, we identify using quantitative RNA-binding assays an N-terminal lysine/arginine-rich motif in WDR33 as a critical determinant of specific AAUAAA motif recognition. Together, these results shed light on the function of CPSF in mediating PAS-dependent RNA cleavage and polyadenylation.

FEM1A, FEM1B, and FEM1C are evolutionarily-conserved VHL-box proteins, the substrate recognition subunits of CUL2-RING E3 ubiquitin ligase complexes. Here, we report that FEM1 proteins are ancient regulators of Stem-Loop Binding Protein (SLBP), a conserved protein that interacts with the stem loop structure located in the 3' end of canonical histone mRNAs and functions in mRNA cleavage, translation and degradation. SLBP levels are highest during S-phase coinciding with histone synthesis. The ubiquitin ligase complex SCF cyclin F targets SLBP for degradation in G2 phase; however, the regulation of SLBP during other stages of the cell cycle is poorly understood. We provide evidence that FEM1A, FEM1B, and FEM1C interact with and mediate the degradation of SLBP. Cyclin F, FEM1A, FEM1B and FEM1C all interact with a region in SLBP's N-terminus using distinct degrons. An SLBP mutant that is unable to interact with all 4 ligases is expressed at higher levels than wild type SLBP and does not oscillate during the cell cycle. We demonstrate that orthologues of SLBP and FEM1 proteins interact in C. elegans and D. melanogaster, suggesting that the pathway is evolutionarily conserved. Furthermore, we show that FEM1 depletion in C. elegans results in the upregulation of SLBP ortholog CDL-1 in oocytes. Notably, cyclin F is absent in flies and worms, suggesting that FEM1 proteins play an important role in SLBP targeting in lower eukaryotes.

SLBP (stem-loop binding protein) is a highly conserved factor necessary for the processing, translation, and degradation of H2AFX and canonical histone mRNAs. We identified the F-box protein cyclin F, a substrate recognition subunit of an SCF (Skp1-Cul1-F-box protein) complex, as the G2 ubiquitin ligase for SLBP. SLBP interacts with cyclin F via an atypical CY motif, and mutation of this motif prevents SLBP degradation in G2. Expression of an SLBP stable mutant results in increased loading of H2AFX mRNA onto polyribosomes, resulting in increased expression of H2A.X (encoded by H2AFX). Upon genotoxic stress in G2, high levels of H2A.X lead to persistent γH2A.X signaling, high levels of H2A.X phosphorylated on Tyr142, high levels of p53, and induction of apoptosis. We propose that cyclin F co-evolved with the appearance of stem-loops in vertebrate H2AFX mRNA to mediate SLBP degradation, thereby limiting H2A.X synthesis and cell death upon genotoxic stress., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In eukaryotes, bulk histone expression occurs in the S phase of the cell cycle. This highly conserved system is crucial for genomic stability and proper gene expression. In metazoans, Stem-loop binding protein (SLBP), which binds to 3' ends of canonical histone mRNAs, is a key factor in histone biosynthesis. SLBP is mainly expressed in S phase and this is a major mechanism to limit bulk histone production to the S phase. At the end of S phase, SLBP is rapidly degraded by proteasome, depending on two phosphorylations on Thr 60 and Thr 61. Previously, we showed that SLBP fragment (aa 51-108) fused to GST, is sufficient to mimic the late S phase (S/G2) degradation of SLBP. Here, using this fusion protein as bait, we performed pull-down experiments and found that DCAF11, which is a substrate receptor of CRL4 complexes, binds to the phosphorylated SLBP fragment. We further confirmed the interaction of full-length SLBP with DCAF11 and Cul4A by co-immunoprecipitation experiments. We also showed that DCAF11 cannot bind to the Thr61/Ala mutant SLBP, which is not degraded at the end of S phase. Using ectopic expression and siRNA experiments, we demonstrated that SLBP expression is inversely correlated with DCAF11 levels, consistent with the model that DCAF11 mediates SLBP degradation. Finally, we found that ectopic expression of the S/G2 stable mutant SLBP (Thr61/Ala) is significantly more toxic to the cells, in comparison to wild type SLBP. Overall, we concluded that CRL4-DCAF11 mediates the degradation of SLBP at the end of S phase and this degradation is essential for the viability of cells.

A rare subset of HIV-1-infected individuals is able to maintain plasma viral load (VL) at low levels without antiretroviral treatment. Identifying the mechanisms underlying this atypical response to infection may lead to therapeutic advances for treating HIV-1. Here, we developed a proteomic analysis to compare peripheral blood cell proteomes in 20 HIV-1-infected individuals who maintained either high or low VL with the aim of identifying host factors that impact HIV-1 replication. We determined that the levels of multiple histone proteins were markedly decreased in cohorts of individuals with high VL. This reduction was correlated with lower levels of stem-loop binding protein (SLBP), which is known to control histone metabolism. Depletion of cellular SLBP increased promoter engagement with the chromatin structures of the host gene high mobility group protein A1 (HMGA1) and viral long terminal repeat (LTR), which led to higher levels of HIV-1 genomic integration and proviral transcription. Further, we determined that TNF-α regulates expression of SLBP and observed that plasma TNF-α levels in HIV-1-infected individuals correlated directly with VL levels and inversely with cellular SLBP levels. Our findings identify SLBP as a potentially important cellular regulator of HIV-1, thereby establishing a link between histone metabolism, inflammation, and HIV-1 infection.

To maintain genome integrity and epigenetic information, mammalian cells must carefully coordinate the supply and deposition of histones during DNA replication. Here we report that the CUL4 E3 ubiquitin ligase complex CRL4(WDR23) directly regulates the stem-loop binding protein (SLBP), which orchestrates the life cycle of histone transcripts including their stability, maturation, and translation. Lack of CRL4(WDR23) activity is characterized by depletion of histones resulting in inhibited DNA replication and a severe slowdown of growth in human cells. Detailed analysis revealed that CRL4(WDR23) is required for efficient histone mRNA 3' end processing to produce mature histone mRNAs for translation. CRL4(WDR23) binds and ubiquitylates SLBP in vitro and in vivo, and this modification activates SLBP function in histone mRNA 3' end processing without affecting its protein levels. Together, these results establish a mechanism by which CUL4 regulates DNA replication and possible additional chromatin transactions by controlling the concerted expression of core histones., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Stem-loop-binding proteins (SLBPs) have been revealed to interact with stem-loop of histones, and oocyte-specific and oocyte-preferential SLBP2 have been identified in vertebrates including Xenopus (S. tropicalis) and B. taurus to play a key role in histone translation regulation, but no oocyte-specific SLBPs have been characterized in fish. Here, we have identified and characterized the first fish oocyte-specific SLBP2 in Carassius gibelio. Its full-length cDNA contains 975 bp ORF encoding 324 amino acids. Firstly, the polyclonal antibody specific to C. gibelio SLBP2 was prepared. Then, RT-PCR analysis and Western blot detection revealed its oocyte-specific and dynamic expression pattern during oogenesis and embryogenesis of C. gibelio. Moreover, in situ hybridization and immunofluorescence localization observed its abundant expression in cortical alveolar stage oocytes and dynamic distribution in different stage oocytes. Altogether, our current data suggest that C. gibelio SLBP2 might play significant role in the early oogenesis and oocyte growth., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Canonical histones are synthesized with a peak in S-phase, whereas histone variants are formed throughout the cell cycle. Unlike messenger RNA (mRNA) for all other genes with a poly(A) tail, canonical histone mRNAs contain a stem-loop structure at their 3'-ends. This stem-loop structure is the binding site for the stem-loop binding protein (SLBP), a protein involved in canonical histone mRNA processing. Recently, we found that arsenic depletes SLBP by enhancing its proteasomal degradation and epigenetically silencing the promoter of the SLBP gene. The loss of SLBP disrupts histone mRNA processing and induces aberrant polyadenylation of canonical histone H3.1 mRNA. Here, we present new data supporting the idea that the lack of SLBP allows the H3.1 mRNA to be polyadenylated using the downstream poly(A) signal. SLBP was also depleted in arsenic-transformed bronchial epithelial cells (BEAS-2B), which led us to hypothesize the involvement of SLBP and polyadenylated H3.1 mRNA in carcinogenesis. Here, for the first time, we report that overexpression of H3.1 polyadenylated mRNA, and knockdown of SLBP enhances anchorage-independent cell growth. A pcDNA-H3.1 vector with a poly(A) signal sequence was stably transfected into BEAS-2B cells. Polyadenylated H3.1 mRNA and exogenous H3.1 protein levels were significantly increased in cells containing the pcDNA-H3.1 vector. A soft agar assay revealed that cells containing the vector formed significantly higher numbers of colonies compared to wild-type cells. Moreover, small hairpin RNA for SLBP (shSLBP) was used to knockdown the expression of SLBP. Cells stably transfected with the shSLBP vector grew significantly more colonies in soft agar than cells transfected with a control vector. These data suggest that upregulation of polyadenylated H3.1 mRNA holds potential as a mechanism to facilitate carcinogenesis by toxicants such as arsenic that depletes SLBP.

The replication-dependent histone genes are the only metazoan genes whose messenger RNA (mRNA) does not terminate with a poly(A) tail at the 3'-end. Instead, the histone mRNAs display a stem-loop structure at their 3'-end. Stem-loop-binding protein (SLBP) binds the stem-loop and regulates canonical histone mRNA metabolism. Here we report that exposure to arsenic, a carcinogenic metal, decreased cellular levels of SLBP by inducing its proteasomal degradation and inhibiting SLBP transcription via epigenetic mechanisms. Notably, arsenic exposure dramatically increased polyadenylation of canonical histone H3.1 mRNA possibly through down-regulation of SLBP expression. The polyadenylated H3.1 mRNA induced by arsenic was not susceptible to normal degradation that occurs at the end of S phase, resulting in continued presence into mitosis, increased total H3.1 mRNA, and increased H3 protein levels. Excess expression of canonical histones have been shown to increase sensitivity to DNA damage as well as increase the frequency of missing chromosomes and induce genomic instability. Thus, polyadenylation of canonical histone mRNA following arsenic exposure may contribute to arsenic-induced carcinogenesis., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Replication-dependent histone mRNAs end with a conserved stem loop that is recognized by stem-loop-binding protein (SLBP). The minimal RNA-processing domain of SLBP is phosphorylated at an internal threonine, and Drosophila SLBP (dSLBP) also is phosphorylated at four serines in its 18-aa C-terminal tail. We show that phosphorylation of dSLBP increases RNA-binding affinity dramatically, and we use structural and biophysical analyses of dSLBP and a crystal structure of human SLBP phosphorylated on the internal threonine to understand the striking improvement in RNA binding. Together these results suggest that, although the C-terminal tail of dSLBP does not contact the RNA, phosphorylation of the tail promotes SLBP conformations competent for RNA binding and thereby appears to reduce the entropic penalty for the association. Increased negative charge in this C-terminal tail balances positively charged residues, allowing a more compact ensemble of structures in the absence of RNA.

The role of replicative DNA polymerases in ensuring genome stability is intensively studied, but the role of other components of the replisome is still not fully understood. One of such component is the GINS complex (comprising the Psf1, Psf2, Psf3 and Sld5 subunits), which participates in both initiation and elongation of DNA replication. Until now, the understanding of the physiological role of GINS mostly originated from biochemical studies. In this article, we present genetic evidence for an essential role of GINS in the maintenance of replication fidelity in Saccharomyces cerevisiae. In our studies we employed the psf1-1 allele (Takayama et al., 2003) and a novel psf1-100 allele isolated in our laboratory. Analysis of the levels and specificity of mutations in the psf1 strains indicates that the destabilization of the GINS complex or its impaired interaction with DNA polymerase epsilon increases the level of spontaneous mutagenesis and the participation of the error-prone DNA polymerase zeta. Additionally, a synergistic mutator effect was found for the defects in Psf1p and in the proofreading activity of Pol epsilon, suggesting that proper functioning of GINS is crucial for facilitating error-free processing of terminal mismatches created by Pol epsilon., (© 2014 John Wiley & Sons Ltd.)

Histone mRNA levels are cell cycle regulated, and the major regulatory steps are at the posttranscriptional level. A major regulatory mechanism is S-phase restriction of Stem-loop binding protein (SLBP) which binds to the 3' end of histone mRNA and participates in multiple steps of histone mRNA metabolism, including 3' end processing, translation and regulation of mRNA stability. SLBP expression is cell cycle regulated without significant change in its mRNA level. SLBP expression is low in G1 until just before S phase where it functions and at the end of S phase SLBP is degraded by proteasome complex depending on phosphorylations on Thr60 and Thr61. Here using synchronized HeLa cells we showed that SLBP production rate is low in early G1 and recovers back to S phase level somewhere between early and mid-G1. Further, we showed that SLBP is unstable in G1 due to proteasome mediated degradation as a novel mechanism to keep SLBP low in G1. Finally, the S/G2 stable mutant form of SLBP is degraded by proteasome in G1, indicating that indicating that the SLBP degradation in G1 is independent of the previously identified SLBP degradation at S/G2. In conclusion, as a mechanism to limit histone production to S phase, SLBP is kept low in G1 phase due to cooperative action of translation regulation and proteasome mediated degradation which is independent of previously known S/G2 degradation., (© 2013 Wiley Periodicals, Inc.)

Transportin 3 (Tnpo3, Transportin-SR2) is implicated in nuclear import of splicing factors and HIV-1 replication. Herein, we show that the majority of cellular Tnpo3 binding partners contain arginine-serine (RS) repeat domains and present crystal structures of human Tnpo3 in its free as well as GTPase Ran- and alternative splicing factor/splicing factor 2 (ASF/SF2)-bound forms. The flexible β-karyopherin fold of Tnpo3 embraces the RNA recognition motif and RS domains of the cargo. A constellation of charged residues on and around the arginine-rich helix of Tnpo3 HEAT repeat 15 engage the phosphorylated RS domain and are critical for the recognition and nuclear import of ASF/SF2. Mutations in the same region of Tnpo3 impair its interaction with the cleavage and polyadenylation specificity factor 6 (CPSF6) and its ability to support HIV-1 replication. Steric incompatibility of the RS domain and RanGTP engagement by Tnpo3 provides the mechanism for cargo release in the nucleus. Our results elucidate the structural bases for nuclear import of splicing factors and the Tnpo3-CPSF6 nexus in HIV-1 biology.

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.

MOZ and MLL encoding a histone acetyltransferase and a histone methyltransferase, respectively, are targets for recurrent chromosomal translocations found in acute myeloblastic or lymphoblastic leukemia. We have previously shown that MOZ and MLL cooperate to activate HOXA9 gene expression in hematopoietic stem/progenitors cells. To dissect the mechanism of action of this complex, we decided to identify new proteins interacting with MOZ. We found that the scaffold protein Symplekin that supports the assembly of polyadenylation machinery was identified by mass spectrometry. Symplekin interacts and co-localizes with both MOZ and MLL in immature hematopoietic cells. Its inhibition leads to a decrease of the HOXA9 protein level but not of Hoxa9 mRNA and to an over-recruitment of MOZ and MLL onto the HOXA9 promoter. Altogether, our results highlight the role of Symplekin in transcription repression involving a regulatory network between MOZ, MLL and Symplekin., (© 2013.)

Animal replication-dependent histone genes produce histone proteins for the packaging of newly replicated genomic DNA. The expression of these histone genes occurs during S phase and is linked to DNA replication via S-phase checkpoints. The histone RNA-binding protein HBP/SLBP (hairpin-binding protein/stem-loop binding protein), an essential regulator of histone gene expression, binds to the conserved hairpin structure located in the 3'UTR (untranslated region) of histone mRNA and participates in histone pre-mRNA processing, translation and histone mRNA degradation. Here, we report the accumulation of alternatively spliced HBP/SLBP transcripts lacking exons 2 and/or 3 in HeLa cells exposed to replication stress. We also detected a shorter HBP/SLBP protein isoform under these conditions that can be accounted for by alternative splicing of HBP/SLBP mRNA. HBP/SLBP mRNA alternative splicing returned to low levels again upon removal of replication stress and was abrogated by caffeine, suggesting the involvement of checkpoint kinases. Analysis of HBP/SLBP cellular localization using GFP (green fluorescent protein) fusion proteins revealed that HBP/SLBP protein and isoforms lacking the domains encoded by exon 2 and exons 2 and 3 were found in the nucleus and cytoplasm, whereas HBP/SLBP lacking the domain encoded by exon 3 was predominantly localised to the nucleus. This isoform lacks the conserved region important for protein-protein interaction with the CTIF [CBP80/20 (cap-binding protein 80/20)]-dependent initiation translation factor and the eIF4E (eukaryotic initiation factor 4E)-dependent translation factor SLIP1/MIF4GD (SLBP-interacting protein 1/MIF4G domain). Consistent with this, we have previously demonstrated that this region is required for the function of HBP/SLBP in cap-dependent translation. In conclusion, alternative splicing allows the synthesis of HBP/SLBP isoforms with different properties that may be important for regulating HBP/SLBP functions during replication stress.

The transcription-export complex (TREX) couples mRNA transcription, processing and nuclear export. We found that CFIm68, a large subunit of a heterotetrameric protein complex mammalian cleavage factor I (CFIm), which is implicated in alternative polyadenylation site choice, co-purified with Thoc5, a component of human TREX. Immunoprecipitation using antibodies against different components of TREX indicated that most likely both complexes interact via an interaction between Thoc5 and CFIm68. Microarray analysis using human HeLa cells revealed that a subset of genes was differentially expressed on Thoc5 knockdown. Notably, the depletion of Thoc5 selectively attenuated the expression of mRNAs polyadenylated at distal, but not proximal, polyadenylation sites, which phenocopied the depletion of CFIm68. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) indicated that CFIm68 preferentially associated with the 5' regions of genes; strikingly, the 5' peak of CFIm68 was significantly and globally reduced on Thoc5 knockdown. We suggest a model in which human Thoc5 controls polyadenylation site choice through the co-transcriptional loading of CFIm68 onto target genes.

The SLIP1-SLBP complex activates translation of replication-dependent histone mRNAs. In this report, we describe how the activity of the SLIP1-SLBP complex is modulated by phosphorylation and oligomerization. Biophysical characterization of the free proteins shows that whereas SLIP1 is a homodimer that does not bind RNA, human SLBP is an intrinsically disordered protein that is phosphorylated at 23 Ser/Thr sites when expressed in a eukaryotic expression system such as baculovirus. The bacterially expressed unphosphorylated SLIP1-SLBP complex forms a 2:2 high-affinity (K(D) < 0.9 nM) heterotetramer that is also incapable of binding histone mRNA. In contrast, phosphorylated SLBP from baculovirus has a weak affinity (K(D) ~3 μM) for SLIP1. Sequential binding of phosphorylated SLBP to the histone mRNA stem-loop motif followed by association with SLIP1 is required to form an "active" ternary complex. Phosphorylation of SLBP at Thr171 promotes dissociation of the heterotetramer to the SLIP1-SLBP heterodimer. Using alanine scanning mutagenesis, we demonstrate that the binding site on SLIP1 for SLBP lies close to the dimer interface. A single-point mutant near the SLIP1 homodimer interface abolished interaction with SLBP in vitro and reduced the abundance of histone mRNA in vivo. On the basis of these biophysical studies, we propose that oligomerization and SLBP phosphorylation may regulate the SLBP-SLIP1 complex in vivo. SLIP1 may act to sequester SLBP in vivo, protecting it from proteolytic degradation as an inactive heterotetramer, or alternatively, formation of the SLIP1-SLBP heterotetramer may facilitate removal of SLBP from the histone mRNA prior to histone mRNA degradation.

TWIST1 is a highly conserved basic helix-loop-helix transcription factor that promotes epithelial-mesenchymal transition (EMT). Its misregulation has been observed in various types of tumors. Using the MCF-10A-series of cell lines that recapitulate the early stages of breast cancer formation and EMT, we found TWIST1 to be upregulated during EMT and downregulated early in carcinogenesis. The TWIST1 3'UTR contains putative regulatory elements, including miRNA target sites and two cytoplasmic polyadenylation elements (CPE). We found that miR-580, CPEB1, and CPEB2 act as negative regulators of TWIST1 expression in a sequence-specific and additive/cooperative manner.

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.

During early embryo development, chromatin packaging is sustained by histones of maternal origin. Most histone messenger RNAs are not polyadenylated, but rather end in an evolutionarily conserved stem-loop that controls RNA processing, nucleocytoplasmic transport, stability, and translation via interactions with a specific protein named stem-loop-binding protein (SLBP). In mouse oocytes, mSLBP is synthesized abundantly during maturation and activates histone translation. In Xenopus, xSLBP is present in stage-VI oocytes, but histone mRNA is protected from premature translation by the oocyte-specific Xenopus SLBP2 (xSLBP2) protein; during maturation xSLBP2 replacement by xSLBP results in histone synthesis. Here, we report the first experimental evidence and characterization of a mammalian SLBP2 ortholog. Bovine bSLBP and bSLBP2 display distinct expression patterns throughout oocyte maturation and pre-implantation embryo development. From the immature oocyte to the morula, bSLBP2 is concentrated in the nucleus, while it is homogeneously distributed throughout the cytoplasm in mature oocytes. A putative SLBP2 gene is conserved in the genome of several mammalian species, and the corresponding transcripts were detected in rat, dog, horse, and pig oocytes. By contrast, a pseudogene is found in mouse, human, and rabbit. Altogether, our data suggest that the availability of histones in oocytes is regulated by an alternative mechanism in bovine and other species as compared to mouse and frog., (Copyright © 2012 Wiley Periodicals, Inc.)

In metazoans, the majority of histone proteins are generated from replication-dependent histone mRNAs. These mRNAs are unique in that they are not polyadenylated but have a stem-loop structure in their 3' untranslated region. An early event in 3' end formation of histone mRNAs is the binding of stem-loop binding protein (SLBP) to the stem-loop structure. Here we provide insight into the mechanism by which SLBP contacts the histone mRNA. There are two binding sites in the SLBP RNA binding domain for the histone mRNA hairpin. The first binding site (Glu129-Val158) consists of a helix-turn-helix motif that likely recognizes the unpaired uridines in the loop of the histone hairpin and, upon binding, destabilizes the first G-C base pair at the base of the stem. The second binding site lies between residues Arg180 and Pro200, which appears to recognize the second G-C base pair from the base of the stem and possibly regions flanking the stem-loop structure. We show that the SLBP-histone mRNA complex is regulated by threonine phosphorylation and proline isomerization in a conserved TPNK sequence that lies between the two binding sites. Threonine phosphorylation increases the affinity of SLBP for histone mRNA by slowing the off rate for complex dissociation, whereas the adjacent proline acts as a critical hinge that may orient the second binding site for formation of a stable SLBP-histone mRNA complex. The nuclear magnetic resonance and kinetic studies presented here provide a framework for understanding how SLBP recognizes histone mRNA and highlight possible structural roles of phosphorylation and proline isomerization in RNA binding proteins in remodeling ribonucleoprotein complexes.

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.

Cleavage/polyadenylation of mRNAs and 3' processing of replication-dependent histone transcripts are both mediated by large complexes that share several protein components. Functional studies of these shared proteins are complicated by the cooperative binding of the individual subunits. For CstF-64, an additional difficulty is that symplekin and CstF-77 bind mutually exclusively to its hinge domain. Here we have identified CstF-64 and symplekin mutants that allowed us to distinguish between these interactions and to elucidate the role of CstF-64 in the two processing reactions. The interaction of CstF-64 with symplekin is limiting for histone RNA 3' processing but relatively unimportant for cleavage/polyadenylation. In contrast, the nuclear accumulation of CstF-64 depends on its binding to CstF-77 and not to symplekin. Moreover, the CstF-64 paralogue CstF-64Tau can compensate for the loss of CstF-64. As CstF-64Tau has a lower affinity for CstF-77 than CstF-64 and is relatively unstable, it is the minor form. However, it may become up-regulated when the CstF-64 level decreases, which has biological implications for spermatogenesis and probably also for other regulatory events. Thus, the interactions between CstF-64/CstF-64Tau and CstF-77 are important for the maintenance of stoichiometric nuclear levels of the CstF complex components and for their intracellular localization, stability, and function.

Phosphorylation of the C-terminal domain (CTD) of RNA polymerase II controls the co-transcriptional assembly of RNA processing and transcription factors. Recruitment relies on conserved CTD-interacting domains (CIDs) that recognize different CTD phosphoisoforms during the transcription cycle, but the molecular basis for their specificity remains unclear. We show that the CIDs of two transcription termination factors, Rtt103 and Pcf11, achieve high affinity and specificity both by specifically recognizing the phosphorylated CTD and by cooperatively binding to neighboring CTD repeats. Single-residue mutations at the protein-protein interface abolish cooperativity and affect recruitment at the 3' end processing site in vivo. We suggest that this cooperativity provides a signal-response mechanism to ensure that its action is confined only to proper polyadenylation sites where Ser2 phosphorylation density is highest.

The heterogeneous nuclear ribonucleoprotein Npl3p of budding yeast is a substrate of arginine methyltransferase Hmt1p, but the role of Hmt1p in regulating Npl3p's functions in transcription antitermination and elongation were unknown. We found that mutants lacking Hmt1p methyltransferase activity exhibit reduced recruitment of Npl3p, but elevated recruitment of a component of mRNA cleavage/termination factor CFI, to the activated GAL10-GAL7 locus. Consistent with this, hmt1 mutants displayed increased termination at the defective gal10-Delta56 terminator. Remarkably, hmt1Delta cells also exhibit diminished recruitment of elongation factor Tho2p and a reduced rate of transcription elongation in vivo. Importantly, the defects in Npl3p and Tho2p recruitment, antitermination and elongation in hmt1Delta cells all were mitigated by substitutions in Npl3p RGG repeats that functionally mimic arginine methylation by Hmt1p. Thus, Hmt1p promotes elongation and suppresses termination at cryptic terminators by methylating RGG repeats in Npl3p. As Hmt1p stimulates dissociation of Tho2p from an Npl3p-mRNP complex, it could act to recycle these elongation and antitermination factors back to sites of ongoing transcription.

Developmentally regulated translation plays a key role in controlling gene expression during oogenesis. In particular, numerous mRNA species are translationally repressed in growing oocytes and become translationally activated during meiotic maturation. While many studies have focused on a U-rich sequence, termed the cytoplasmic polyadenylation element (CPE), located in the 3'-untranslated region (UTR) and the CPE-binding protein (CPEB) 1, multiple mechanisms likely contribute to translational control in oocytes. The stem-loop-binding protein (SLBP) is expressed in growing oocytes, where it is required for the accumulation of nonpolyadenylated histone mRNAs, and then accumulates substantially during meiotic maturation. We report that, in immature oocytes, Slbp mRNA carries a short poly(A) tail, and is weakly translated, and that a CPE-like sequence in the 3'-UTR is required to maintain this low activity. During maturation, Slbp mRNA becomes polyadenylated and translationally activated. Unexpectedly, proteasomal activity is required both to initiate and to sustain translational activation. This proteasomal activity is not required for the polyadenylation of Slbp mRNA during early maturation; however, it is required for a subsequent deadenylation of the mRNA that occurs during late maturation. Moreover, although CPEB1 is degraded during maturation, inhibiting its degradation by blocking mitogen-activated protein kinase 1/3 activity does not prevent the accumulation of SLBP, indicating that CPEB1 is not the protein whose degradation is required for translational activation of Slbp mRNA. These results identify a new role for proteasomal activity in initiating and sustaining translational activation during meiotic maturation.

Metazoan histone mRNAs are unique: their pre-mRNAs contain no introns, and the mRNAs are not polyadenylated, ending instead in a conserved stem-loop structure. In Drosophila, canonical poly(A) signals are located downstream of the normal cleavage site of each histone gene and are utilized when histone 3' end formation is inhibited. Here we define a subcomplex of poly(A) factors that are required for histone pre-mRNA processing. We demonstrate that Symplekin, CPSF73, and CPSF100 are present in a stable complex and interact with histone-specific processing factors. We use chromatin immunoprecipitation to show that Symplekin and CPSF73, but not CstF50, cotranscriptionally associate with histone genes. Depletion of SLBP recruits CstF50 to histone genes. Knockdown of CPSF160 or CstF64 downregulates Symplekin but does not affect histone pre-mRNA processing or association of Symplekin with the histone locus. These results suggest that a common core cleavage factor is required for processing of histone and polyadenylated pre-mRNAs.

Oocytes accumulate mRNAs and proteins that direct early embryonic development. Although subsequent development requires the timely degradation of these maternal products, little is known of the underlying mechanisms. The stem-loop-binding protein (SLBP), which regulates the stability and translation of mRNAs encoding histones and is synthesized during S-phase and degraded during G2 in somatic cells, accumulates during oogenesis. Maternal SLBP is required for mouse embryos to develop beyond the 2-cell stage, but must be degraded to allow the cell-cycle-regulated expression of somatic cells to be established. We report that the quantity of maternal SLBP changes little following fertilization until 44-52 h post-hCG, corresponding to mid-/late G2 of the 2-cell stage, when it decreases by 75%. Efficient degradation requires two pathways. The first requires activity of cyclin-dependent kinases (cdk) and embryonic transcription, preferentially targets nuclear SLBP, and likely corresponds to the pathway that degrades SLBP at G2 in somatic cells. The second does not require cdk activity or transcription and becomes active at 44-52 h post-hCG independently of cell-cycle progression to mid-/late G2, but is not solely regulated by the time elapsed since hCG injection. Thus, the co-ordinated activity of two separately regulated pathways eliminates maternally encoded SLBP from early mouse embryos.

Histone mRNAs are the only eukaryotic cellular mRNAs that are not polyadenylated. Synthesis of mature histone mRNA requires only a single processing reaction: an endonucleolytic cleavage between a conserved stem-loop and a purine-rich downstream element to form the 3' end. The stem-loop binding protein (SLBP) is required for processing, and following processing, histone mRNA is transported to the cytoplasm, where SLBP participates in translation of the histone mRNA and is also involved in regulation of histone mRNA degradation. Here we present an analysis of histone mRNA metabolism in cells with highly reduced levels of SLBP using RNA interference. Knocking down SLBP in U2OS cells results in a reduction in the rate of cell growth and an accumulation of cells in S-phase. Surprisingly, there is only a modest (twofold) decrease in histone mRNA levels. Much of histone mRNA in the SLBP knockdown cells is properly processed but is retained in the nucleus. The processed histone mRNA in SLBP knockdown cells is not rapidly degraded when DNA replication is inhibited. These results suggest a previously undescribed role for SLBP in histone mRNA export.

We investigated recruitment of the yeast mRNA export factor Yra1 to the transcription elongation complex (TEC). Previously, the Sub2 helicase subunit of TREX was proposed to recruit Yra1. We report that Sub2 is dispensable for Yra1 recruitment, but the cleavage/polyadenylation factor, CF1A, is required. Yra1 binds directly to the Zn finger/Clp1 region of Pcf11, the pol II CTD-binding subunit of CF1A, and this interaction is conserved between their human homologs. Tethering of Pcf11 to nascent mRNA is sufficient to enhance Yra1 recruitment. Interaction with Pcf11 can therefore explain Yra1 binding to the TEC independently of Sub2. We propose that after initially binding to Pcf11, Yra1 is transferred to Sub2. Consistent with this idea, Pcf11 binds the same regions of Yra1 that also contact Sub2, indicating a mutually exclusive interaction. These results suggest a mechanism for cotranscriptional assembly of the export competent mRNP and for coordinating export with 3' end processing.

The production of a functional mRNA is regulated at every step of transcription. An area not well-understood is the transition of RNA polymerase II from elongation to termination. The S. cerevisiae SR-like protein Npl3 functions to negatively regulate transcription termination by antagonizing the binding of polyA/termination proteins to the mRNA. In this study, Npl3 is shown to interact with the CTD and have a direct stimulatory effect on the elongation activity of the polymerase. The interaction is inhibited by phosphorylation of Npl3. In addition, Casein Kinase 2 was found to be required for the phosphorylation of Npl3 and affect its ability to compete against Rna15 (Cleavage Factor I) for binding to polyA signals. Our results suggest that phosphorylation of Npl3 promotes its dissociation from the mRNA/RNAP II, and contributes to the association of the polyA/termination factor Rna15. This work defines a novel role for Npl3 in elongation and its regulation by phosphorylation.

Histone mRNA levels are cell cycle regulated, and a major regulatory mechanism is restriction of stem-loop binding protein (SLBP) to S phase. Degradation of SLBP at the end of S phase results in cessation of histone mRNA biosynthesis, preventing accumulation of histone mRNA until SLBP is synthesized just before entry into the next S phase. Degradation of SLBP requires an SFTTP (58 to 62) and KRKL (95 to 98) sequence, which is a putative cyclin binding site. A fusion protein with the 58-amino-acid sequence of SLBP (amino acids 51 to 108) fused to glutathione S-transferase (GST) is sufficient to mimic SLBP degradation at late S phase. Using GST-SLBP fusion proteins as a substrate, we show that cyclin A/Cdk1 phosphorylates Thr61. Furthermore, knockdown of Cdk1 by RNA interference stabilizes SLBP at the end of S phase. Phosphorylation of Thr61 is necessary for subsequent phosphorylation of Thr60 by CK2 in vitro. Inhibitors of CK2 also prevent degradation of SLBP at the end of S phase. Thus, phosphorylation of Thr61 by cyclin A/Cdk1 primes phosphorylation of Thr60 by CK2 and is responsible for initiating SLBP degradation. We conclude that the increase in cyclin A/Cdk1 activity at the end of S phase triggers degradation of SLBP at S/G(2).

The Paf1 complex (Paf1, Ctr9, Cdc73, Rtf1, and Leo1) is normally associated with RNA polymerase II (Pol II) throughout the transcription cycle. However, the loss of either Rtf1 or Cdc73 results in the detachment of the Paf1 complex from Pol II and the chromatin form of actively transcribed genes. Using functionally tagged forms of the Paf1 complex factors, we have determined that, except for the more loosely associated Rtf1, the remaining components stay stably associated with one another in an RNase-resistant complex after dissociation from Pol II and chromatin. The loss of Paf1, Ctr9, or to a lesser extent Cdc73 or Rtf1 results in reduced levels of serine 2 phosphorylation of the Pol II C-terminal domain and in increased read through of the MAK21 polyadenylation site. We found that the cleavage and polyadenylation factor Cft1 requires the Pol II-associated form of the Paf1 complex for full levels of interaction with the serine 5-phosphorylated form of Pol II. When the Paf1 complex is dissociated from Pol II, a direct interaction between Cft1 and the Paf1 complex can be detected. These results are consistent with the Paf1 complex providing a point of contact for recruitment of 3'-end processing factors at an early point in the transcription cycle. The lack of this connection helps to explain the defects in 3'-end formation observed in the absence of Paf1.

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.

Growing oocytes accumulate mRNAs and proteins that support early embryogenesis. Among the most abundant of these maternal factors are the histones. Histone mRNA accumulation and translation are mainly restricted to S-phase in somatic cells, and the mechanism by which oocytes produce histones is unknown. In somatic cells, replication-dependent histone synthesis requires the stem-loop binding protein (SLBP). SLBP is expressed during S-phase, binds to the 3'-untranslated region of non-polyadenylated transcripts encoding the histones, and is required for their stabilization and translation. SLBP is expressed in oocytes of several species, suggesting a role in histone synthesis. To test this, we generated transgenic mice whose oocytes lack SLBP. mRNAs encoding histones H3 and H4 failed to accumulate in these oocytes. Unexpectedly, mRNAs encoding H2A and H2B were little affected. Embryos derived from SLBP-depleted oocytes reached the 2-cell stage, but most then became arrested. Histones H3 and H4, but not H2A or H2B, were substantially reduced in these embryos. The embryos also expressed high levels of gamma H2A.X. Injection of histones into SLBP-depleted embryos rescued them from developmental arrest. Thus, SLBP is an essential component of the mechanism by which growing oocytes of the mouse accumulate the histones that support early embryonic development.

Multiple steps in mRNA processing and transcription are coupled. Notably, the processing of mRNA 3' ends is linked to transcription termination by RNA polymerase II. Previously, we found that the yeast hnRNP protein Npl3 can negatively regulate 3' end mRNA formation and termination at the GAL1 gene. Here we show that overexpression of the Hrp1 or Rna14 subunits of the CF IA polyadenylation factor increases recognition of a weakened polyadenylation site. Genetic interactions of mutant alleles of NPL3 or HRP1 with RNA15 also indicate antagonism between these factors. Npl3 competes with Rna15 for binding to a polyadenylation precursor and inhibits cleavage and polyadenylation in vitro. These results suggest that an important function of hnRNP proteins is to ensure the fidelity of mRNA processing. Our results support a model in which balanced competition of Npl3 with mRNA processing factors may promote recognition of proper polyadenylation sites while suppressing cryptic sites.

Negative elongation factor (NELF) is a four subunit transcription elongation factor that has been implicated in numerous diseases ranging from neurological disorders to cancer. Here we show that NELF interacts with the nuclear cap binding complex (CBC), a multifunctional factor that plays important roles in several mRNA processing steps, and the two factors together participate in the 3' end processing of replication-dependent histone mRNAs, most likely through association with the histone stem-loop binding protein (SLBP). Strikingly, absence of NELF and CBC causes aberrant production of polyadenylated histone mRNAs. Moreover, NELF is physically associated with histone gene loci and forms distinct intranuclear foci that we call NELF bodies, which often overlap with Cajal bodies and cleavage bodies. Our results point to a surprising role of NELF in the 3' end processing of histone mRNAs and also suggest that NELF is a new factor that coordinates different mRNA processing steps during transcription.

The protein factor U2 snRNP Auxiliary Factor (U2AF) 65 is an essential component required for splicing and involved in the coupling of splicing and 3' end processing of vertebrate pre-mRNAs. Here we have addressed the mechanisms by which U2AF 65 stimulates pre-mRNA 3' end processing. We identify an arginine/serine-rich region of U2AF 65 that mediates an interaction with an RS-like alternating charge domain of the 59 kDa subunit of the human cleavage factor I (CF I(m)), an essential 3' processing factor that functions at an early step in the recognition of the 3' end processing signal. Tethered functional analysis shows that the U2AF 65/CF I(m) 59 interaction stimulates in vitro 3' end cleavage and polyadenylation. These results therefore uncover a direct role of the U2AF 65/CF I(m) 59 interaction in the functional coordination of splicing and 3' end processing.

The replication-dependent histone mRNAs are cell-cycle-regulated and expressed only during S phase. In contrast to all other eukaryotic mRNAs, the histone mRNAs end in a highly conserved 16-nucleotide stem-loop rather than a poly(A) tail. The stem-loop is necessary and sufficient for the post-transcriptional regulation of histone mRNA during the cell cycle. The histone mRNA 3' stem-loop is bound by the stem-loop binding protein (SLBP) that is involved in pre-mRNA processing, translation, and stability of histone mRNA. Immunoprecipitation (IP) of RNA-binding proteins (RBPs) followed by microarray analysis has been used to identify the targets of RNA-binding proteins. This method is sometimes referred to as RIP-Chip (RNA IP followed by microarray analysis). Here we introduce a variation on the RIP-Chip method that uses a recombinant RBP to identify mRNA targets in a pool of total RNA; we call this method recombinant, or rRIP-Chip. Using this method, we show that recombinant SLBP binds exclusively to all five classes of histone mRNA. We also analyze the messages bound to the endogenous SLBP on polyribosomes by immunoprecipitation. We use two different microarray platforms to identify enriched mRNAs. Both platforms demonstrate remarkable specificity and consistency of results. Our data suggest that the replication-dependent histone mRNAs are likely to be the sole target of SLBP.

The stem-loop-binding protein (SLBP) is involved in multiple aspects of histone mRNA metabolism. To characterize the modification status and sites of SLBP, we combined mass spectrometric bottom-up (analysis of peptides) and top-down (analysis of intact proteins) proteomic approaches. Drosophilia SLBP is heavily phosphorylated, containing up to seven phosphoryl groups. Accurate M(r) determination by Fourier transform ion cyclotron resonance (FTICR)-MS and FTICR-MS top-down experiments using a variety of dissociation techniques show there is removal of the initiator methionine and acetylation of the N terminus in the baculovirus-expressed protein, and that T230 is stoichiometrically phosphorylated. T230 is highly conserved; we have determined that this site is also completely phosphorylated in baculovirus-expressed mammalian SLBP and extensively phosphorylated in both Drosophila and mammalian cultured cells. Removal of the phosphoryl group from T230 by either dephosphorylation or mutation results in a 7-fold reduction in the affinity of SLBP for the stem-loop RNA.

In metazoans, cell-cycle-dependent histones are produced from poly(A)-lacking mRNAs. The 3' end of histone mRNAs is formed by an endonucleolytic cleavage of longer precursors between a conserved stem-loop structure and a purine-rich histone downstream element (HDE). The cleavage requires at least two trans-acting factors: the stem-loop binding protein (SLBP), which binds to the stem-loop and the U7 snRNP, which anchors to histone pre-mRNAs by annealing to the HDE. Using RNA structure-probing techniques, we determined the secondary structure of the 3'-untranslated region (3'-UTR) of mouse histone pre-mRNAs H4-12, H1t and H2a-614. Surprisingly, the HDE is embedded in hairpin structures and is therefore not easily accessible for U7 snRNP anchoring. Probing of the 3'-UTR in complex with SLBP revealed structural rearrangements leading to an overall opening of the structure especially at the level of the HDE. Electrophoretic mobility shift assays demonstrated that the SLBP-induced opening of HDE actually facilitates U7 snRNA anchoring on the histone H4-12 pre-mRNAs 3' end. These results suggest that initial binding of the SLBP functions in making the HDE more accessible for U7 snRNA anchoring.