A Complex Containing the CPSF73 Endonuclease and Other Polyadenylation Factors Associates with U7 snRNP and Is Recruited to Histone Pre-mRNA for 3′-End Processing

Animal replication-dependent histone pre-mRNAs are processed at the 3′ end by endonucleolytic cleavage that is not followed by polyadenylation. The cleavage reaction is catalyzed by CPSF73 and depends on the U7 snRNP and its integral component, Lsm11. A critical role is also played by the 220-kDa protein FLASH, which interacts with Lsm11. Here we demonstrate that the N-terminal regions of these two proteins form a platform that tightly interacts with a unique combination of polyadenylation factors: symplekin, CstF64, and all CPSF subunits, including the endonuclease CPSF73. The interaction is inhibited by alterations in each component of the FLASH/Lsm11 complex, including point mutations in FLASH that are detrimental for processing. The same polyadenylation factors are associated with the endogenous U7 snRNP and are recruited in a U7-dependent manner to histone pre-mRNA. Collectively, our studies identify the molecular mechanism that recruits the CPSF73 endonuclease to histone pre-mRNAs, reveal an unexpected complexity of the U7 snRNP, and suggest that in animal cells polyadenylation factors assemble into two alternative complexes—one specifically crafted to generate polyadenylated mRNAs and the other to generate nonpolyadenylated histone mRNAs that end with the stem-loop.     

The 68?kDa subunit of mammalian cleavage factor I interacts with the U7 small nuclear ribonucleoprotein and participates in 3′-end processing of animal histone mRNAs

Metazoan replication-dependent histone pre-mRNAs undergo a unique 3′-cleavage reaction which does not result in mRNA polyadenylation. Although the cleavage site is defined by histone-specific factors (hairpin binding protein, a 100-kDa zinc-finger protein and the U7 snRNP), a large complex consisting of cleavage/polyadenylation specificity factor, two subunits of cleavage stimulation factor and symplekin acts as the effector of RNA cleavage. Here, we report that yet another protein involved in cleavage/polyadenylation, mammalian cleavage factor I 68-kDa subunit (CF I_m68), participates in histone RNA 3′-end processing. CF I_m68 was found in a highly purified U7 snRNP preparation. Its interaction with the U7 snRNP depends on the N-terminus of the U7 snRNP protein Lsm11, known to be important for histone RNA processing. In vivo, both depletion and overexpression of CF I_m68 cause significant decreases in processing efficiency. In vitro 3′-end processing is slightly stimulated by the addition of low amounts of CF I_m68, but inhibited by high amounts or by anti-CF I_m68 antibody. Finally, immunoprecipitation of CF I_m68 results in a strong enrichment of histone pre-mRNAs. In contrast, the small CF I_m subunit, CF I_m25, does not appear to be involved in histone RNA processing.     

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.     

CstF-64 supports pluripotency and regulates cell cycle progression in embryonic stem cells through histone 3′ end processing

Embryonic stem cells (ESCs) exhibit a unique cell cycle with a shortened G₁ phase that supports their pluripotency, while apparently buffering them against pro-differentiation stimuli. In ESCs, expression of replication-dependent histones is a main component of this abbreviated G₁ phase, although the details of this mechanism are not well understood. Similarly, the role of 3′ end processing in regulation of ESC pluripotency and cell cycle is poorly understood. To better understand these processes, we examined mouse ESCs that lack the 3′ end-processing factor CstF-64. These ESCs display slower growth, loss of pluripotency and a lengthened G₁ phase, correlating with increased polyadenylation of histone mRNAs. Interestingly, these ESCs also express the τCstF-64 paralog of CstF-64. However, τCstF-64 only partially compensates for lost CstF-64 function, despite being recruited to the histone mRNA 3′ end-processing complex. Reduction of τCstF-64 in CstF-64-deficient ESCs results in even greater levels of histone mRNA polyadenylation, suggesting that both CstF-64 and τCstF-64 function to inhibit polyadenylation of histone mRNAs. These results suggest that CstF-64 plays a key role in modulating the cell cycle in ESCs while simultaneously controlling histone mRNA 3′ end processing.     

Structure–function analysis and genetic interactions of the Yhc1, SmD3, SmB,and Snp1 subunits of yeast U1 snRNP and genetic interactions of SmD3 with U2 snRNP subunit Lea1

Yhc1 and U1-C are essential subunits of the yeast and human U1 snRNP, respectively, that stabilize the duplex formed by U1 snRNA at the pre-mRNA 5′ splice site (5′SS). Mutational analysis of Yhc1, guided by the human U1 snRNP crystal structure, highlighted the importance of Val20 and Ser19 at the RNA interface. Though benign on its own, V20A was lethal in the absence of branchpoint-binding complex subunit Mud2 and caused a severe growth defect in the absence of U1 subunit Nam8. S19A caused a severe defect with mud2▵. Essential DEAD-box ATPase Prp28 was bypassed by mutations of Yhc1 Val20 and Ser19, consistent with destabilization of U1•5′SS interaction. We extended the genetic analysis to SmD3, which interacts with U1-C/Yhc1 in U1 snRNP, and to SmB, its neighbor in the Sm ring. Whereas mutations of the interface of SmD3, SmB, and U1-C/Yhc1 with U1-70K/Snp1, or deletion of the interacting Snp1 N-terminal peptide, had no growth effect, they elicited synthetic defects in the absence of U1 subunit Mud1. Mutagenesis of the RNA-binding triad of SmD3 (Ser-Asn-Arg) and SmB (His-Asn-Arg) provided insights to built-in redundancies of the Sm ring, whereby no individual side-chain was essential, but simultaneous mutations of Asn or Arg residues in SmD3 and SmB were lethal. Asn-to-Ala mutations SmB and SmD3 caused synthetic defects in the absence of Mud1 or Mud2. All three RNA site mutations of SmD3 were lethal in cells lacking the U2 snRNP subunit Lea1. Benign C-terminal truncations of SmD3 were dead in the absence of Mud2 or Lea1 and barely viable in the absence of Nam8 or Mud1. In contrast, SMD3-E35A uniquely suppressed the temperature-sensitivity of lea1▵.     

A single-base deletion in soybean flavonoid 3′-hydroxylase gene is associated with gray pubescence color

The T locus of soybean (Glycine max (L.) Merr.) controls pubescence and seed coat color and is presumed to encode flavonoid 3-hydroxylase (F3H). The dominant T and the recessive t allele of the locus produce brown and gray pubescence, respectively. PCR primers were constructed based on the sequence of a soybean EST clone homologous to the F3H gene. A putative full-length cDNA, sf3h1 was isolated by 3 and 5 RACE. Sequence analysis revealed that sf3h1 consists of 1690 nucleotides encoding 513 amino acids. It had 68% and 66% homology with corresponding F3H protein sequences of petunia and Arabidopsis, respectively. A conserved amino acid sequence of F3H proteins, GGEK, was found in the deduced polypeptide. Sequence analysis of the gene from a pair of near-isogenic lines for T, To7B (TT, brown) and To7G (tt, gray) revealed that they differed by a single C deletion in the coding region of To7G. The deletion changed the subsequent reading frame resulting in a truncated polypeptide lacking the GGEK consensus sequence and the heme-binding domain. Genomic Southern analysis probed by sf3h1 revealed restriction fragment length polymorphisms between cultivars with different pubescence color. Further, sf3h1 was mapped at the same position with T locus on LG3(c2). PCR-RFLP analysis was performed to detect the single-base deletion. To7B and three cultivars with brown pubescence exhibited shorter fragments, while To7G and three cultivars with gray pubescence had longer fragments due to the single-base deletion. The PCR-RFLP marker co-segregated with genotypes at the Tlocus in a F₂ population segregating for the T locus. The above results strongly suggest that sf3h1 represents the T gene of soybean responsible for pubescence color and that the single-base deletion may be responsible for gray pubescence color.     

Changes in histone phosphorylation and associated early metabolic events in pig lymphocyte cultures transformed by phytohaemagglutinin or 6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate

1. Pig lymphocytes were transformed by dibutyryl cyclic AMP (6-N,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate) at concentrations of 0.01-0.1mum. The pattern of incorporation of label from [5-(3)H]uridine and [6-(3)H]thymidine into RNA and DNA respectively was identical with that obtained with unpurified phytohaemagglutinin. 2. Chlorpromazine (0.1mum) prevented the stimulation of [5-(3)H]uridine incorporation into RNA by phytohaemagglutinin, but only slightly lowered the lymphocyte response to dibutyryl cyclic AMP. 3. An increase in the size and specific radioactivity of the intracellular P(i) pool was found immediately after stimulation by both phytohaemagglutinin and dibutyryl cyclic AMP. This was followed after some 30min by a rise in the specific radioactivity and concentration of ATP. 4. There was an immediate increase in the specific radioactivity of phosphate groups of histones; by about 45min after stimulation only the histones remaining after extraction of histone fraction F1 continued to incorporate (32)P from [(32)P]P(i). 5. Histone kinase activity increased in the first 30min after stimulation; subsequently histone F1 kinase activity decreased, but activity with the other histones as substrate continued to increase for a further 30min. Kinase activation was effected by cyclic AMP (adenosine 3':5'-cyclic monophosphate). 6. Histone phosphatase activity behaved similarly to that of the kinase.     

A 5′UTR SNP of GHRHR locus is associated with body weight and average daily gain in Chinese cattle

Growth hormone-releasing hormone receptor (GHRHR) has important functions in the regulation of the growth hormone axis and the development and proliferation of pituitary somatotropes. Moreover, some mutations in mouse GHRHR can induce the dwarfism. The objective of this paper is to reveal the association of GHRHR with growth traits in three Chinese cattle breeds, including Nanyang cattle (NY, 220), Qinchuan cattle (QC, 114), and Jiaxian cattle (JX, 142). A novel single nucleotide polymorphism (NM_181020:c.102C>T) in 5′UTR of GHRHR was identified using PCR–SSCP and DNA sequencing. The frequency of NM_181020:c.102C allele ranged from 0.926 to 0.956. We found that the locus was significantly associated with NY cattle’s body weight (BW) of 6?months, with average daily gain (ADG) of 0–6?months, and as well as with ADG of 6–12?months (p?<?0.05). The data suggested that the polymorphism (NM_181020:c.102C>T) of the GHRHR could be a molecular marker candidate for breeding of NY cattle in favor of BW.     

Evidence that a polymorphism within the 3′UTR of glutathione peroxidase 4 is functional and is associated with susceptibility to colorectal cancer

Low selenium (Se) status has been associated with increased risk of colorectal cancer (CRC). Se is present as the amino acid selenocysteine in selenoproteins, such as the glutathione peroxidases. Se incorporation requires specific RNA structures in the 3' untranslated region (3'UTR) of the selenoprotein mRNAs. A single nucleotide polymorphism (SNP) occurs at nucleotide 718 (within the 3'UTR) in the glutathione peroxidase 4 gene. In the present study, Caco-2 cells were transfected with constructs in which type 1 iodothyronine deiodinase coding region was linked to the GPx4 3'UTR with either C or T variant at position 718. Higher reporter activity was observed in cells expressing the C variant compared to those expressing the T variant, under either Se-adequate or Se-deficient conditions. In addition, a disease association study was carried out in cohorts of patients with either adenomatous polyps, colorectal adenocarcinomas and in healthy controls. A higher proportion of individuals with CC genotype at the GPx4 T/C 718 SNP was present in the cancer group, but not in the polyp group, compared with the control group (P < 0.05). The present data demonstrate the functionality of the GPx4 T/C 718 SNP and suggest that T genotype is associated with lower risk of CRC.     

Overlapping and distinct functions of CstF64 and CstF64τ in mammalian mRNA 3′ processing

mRNA 3′ processing is dynamically regulated spatially and temporally. However, the underlying mechanisms remain poorly understood. CstF64τ is a paralog of the general mRNA 3′ processing factor, CstF64, and has been implicated in mediating testis-specific mRNA alternative polyadenylation (APA). However, the functions of CstF64τ in mRNA 3′ processing have not been systematically investigated. We carried out a comprehensive characterization of CstF64τ and compared its properties to those of CstF64. In contrast to previous reports, we found that both CstF64 and CstF64τ are widely expressed in mammalian tissues, and their protein levels display tissue-specific variations. We further demonstrated that CstF64 and CstF64τ have highly similar RNA-binding specificities both in vitro and in vivo. CstF64 and CstF64τ modulate one another''s expression and play overlapping as well as distinct roles in regulating global APA profiles. Interestingly, protein interactome analyses revealed key differences between CstF64 and CstF64τ, including their interactions with another mRNA 3′ processing factor, symplekin. Together, our study of CstF64 and CstF64τ revealed both functional overlap and specificity of these two important mRNA 3′ processing factors and provided new insights into the regulatory mechanisms of mRNA 3′ processing.     

One functional variant in the 3′-untranslated region of TLR4 is associated with the elevated risk of ventilator-associated pneumonia in the patients with chronic obstructive pulmonary disease

The aim of this study was to identify the association polymorphism (rs11536889) in the 3′-untranslated region (3′-UTR) of Toll-like receptors 4 (TLR4) and the risk for ventilator-associated pneumonia (VAP). miRNA database online and luciferase assays were used to validate TLR4 as the target gene of miR-1236. Enzyme-linked immunosorbent assay analysis and western blot were used to analyze the level of TLR4 in different genotype groups. In the present study, miR-1236 was predicted to bind to the rs11536889 G allele rather than the rs11536889 C allele, which was further confirmed by the luciferase activity suppressed by a fragment of 3′-UTR containing the rs11536889 G allele induced by lipopolysaccharide (LPS) and interleukin-6 (IL-6). Bronchial epithelial cells isolated from participants genotyped as GG, GC, and CC, with no remarkable difference in TLR4 messenger RNA (mRNA) levels were observed among these genotype groups. After stimulating by LPS, a TLR4 ligand, the CC-genotyped cells expressed higher levels of IL-8, IL-6, and tumor necrosis factor alpha (TNF-α) on their surfaces than cells with the other genotypes. Finally, the western blot analysis results showed that the expression level of IL-8, IL-6, and TNF-α protein was much higher in the CC group than the GC and GG groups subsequent to stimulation by LPS, and the IL-8, IL-6, and TNF-α protein levels in the GC were grouped much lower compared with the GG group. These findings indicated the regulatory association of miR-1236 with TLR4 and the abnormal expression of TLR4 caused by the presence of rs11536889 in the 3′-UTR of mRNA, which interfere with its interaction with the miR-1236, contributing to the risk of VAP.