Replication-associated histone genes encode the only metazoan mRNAs that lack polyA tails, ending instead in a conserved 26-nt sequence that forms a stem-loop. Most of the regulation of mammalian histone mRNA is posttranscriptional and mediated by this unique 3 end. Stem-loop-binding protein (SLBP) binds to the histone mRNA 3 end and is thought to participate in all aspects of histone mRNA metabolism, including cell cycle regulation. To examine SLBP function genetically, we have cloned the gene encoding Drosophila SLBP (dSLBP) by a yeast three-hybrid method and have isolated mutations in dSLBP. dSLBP function is required both zygotically and maternally. Strong dSLBP alleles cause zygotic lethality late in development and result in production of stable histone mRNA that accumulates in nonreplicating cells. These histone mRNAs are cytoplasmic and have polyadenylated 3 ends like other polymerase II transcripts. Hypomorphic dSLBP alleles support zygotic development but cause female sterility. Eggs from these females contain dramatically reduced levels of histone mRNA, and mutant embryos are not able to complete the syncytial embryonic cycles. This is in part because of a failure of chromosome condensation at mitosis that blocks normal anaphase. These data demonstrate that dSLBP is required in vivo for 3 end processing of histone pre-mRNA, and that this is an essential function for development. Moreover, dSLBP-dependent processing plays an important role in coupling histone mRNA production with the cell cycle.

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.T he vast majority of eukaryotic pre-mRNAs are processed at the 3= end by cleavage coupled to polyadenylation (1-4). In this reaction, pre-mRNAs are cleaved 15 to 30 nucleotides after the highly conserved AAUAAA sequences and the upstream cleavage product is extended by addition of a poly(A) tail. Cleavage coupled to polyadenylation is carried out by a macromolecular machinery consisting of multiple proteins that assemble into at least four separate subcomplexes or factors. The AAUAAA sequence is recognized by cleavage and polyadenylation specificity factor (CPSF), which contains CPSF160, CPSF100, CPSF73, CPSF30, Fip1 (5), and the recently identified WDR33 (6). CPSF160 directly contacts the AAUAAA hexanucleotide, whereas CPSF73 is the endonuclease that catalyzes the cleavage reaction (7). Cleavage stimulation factor (CstF), consisting of CstF77, CstF64, and CstF50, recognizes the GU-rich sequence located downstream of the cleavage site. CstF64 makes direct contacts with this sequence and also interacts with CstF77, which in turn interacts with CstF50 (8). 3=-end processing by cleavage and polyadenylation additionally requires cleavage factor (CF) I m , consisting of 25-kDa and 68-kDa subunits (9), and cleavage factor II m containing at least two subunits, Pcf11 and Clp1 (2, 10). Individual components of the cleavage and polyadenylation machinery are connected with each other through a dense network of protein-protein interactions that stabilizes the entire complex and juxtaposes CPSF73 with the cleavage site. An important role in forming this network is played by symplekin, a protein that interacts with a number of polyadenylation factors and likely functions as a scaffold in 3=-end processing (8,11,12) and other processes, including cytoplasmi...

The stem-loop binding protein (SLBP) is the posttranscriptional regulator of histone mRNA in metazoan cells. SLBP binds histone pre-mRNAs and facilitates 3-end processing by promoting stable association of U7 snRNP with the pre-mRNA. To identify other factors involved in histone pre-mRNA processing, we used a modified yeast two-hybrid assay in which SLBP and its RNA target were coexpressed as bait. A novel zinc finger protein, hZFP100, which interacts with the SLBP/RNA complex but not with free SLBP, was cloned. The interaction requires regions of SLBP that are important for histone pre-mRNA processing. Antibodies to hZFP100 precipitate U7 snRNA, and expression of hZFP100 in Xenopus oocytes stimulates processing of histone pre-mRNA, showing that hZFP100 is a component of the processing machinery.

The present study aims to improve the biological activities of luteolin by complexation with manganese(II).UV-visible spectroscopy, infrared spectroscopy, thermogravimetric analysis and elemental analysis were adopted to assess the relevant interaction of luteolin and manganese(II) ions and the chelation sites. The antioxidant activity, hypoglycemic effect and antimicrobial activity of luteolin-manganese(II) complex with respect to its parent luteolin and the inhibition effect of which on xanthine oxidase were investigated and compared. The spectroscopic data indicated that luteolin reacts with manganese(II) cations through the chelation sites of 5-hydroxy and 4-carbonyl in two luteolin molecules. Antioxidant and antibacterial activity were enhanced after the complexation of manganese(II) cations with luteolin. An inhibition effect assay found that luteolin and luteolin-manganese(II) complex reversibly inhibited xanthine oxidase in a competitive manner. Luteolin-manganese(II) complex had a more remarkable hypoglycemic effect than luteolin by increasing the glucose consumption in liver tissue.

Cinnamoyl-CoA reductase (CCR) is the first enzyme in the monolignol-specific branch of the lignin biosynthetic pathway. In this research, three sorghum CCR genes including SbCCR1, SbCCR2-1 and SbCCR2-2 were cloned and characterized. Analyses of the structure and phylogeny of the three CCR genes showed evolutionary conservation of the functional domains and divergence of function. Transient expression assays in Nicotiana benthamiana leaves demonstrated that the three CCR proteins were localized in the cytoplasm. The expression analysis showed that the three CCR genes were induced by drought. But in 48 h, the expression levels of SbCCR1 and SbCCR2-2 did not differ between CK and the drought treatment; while the expression level of SbCCR2-1 in the drought treatment was higher than in CK. The expression of the SbCCR1 and SbCCR2-1 genes was not induced by sorghum aphid [Melanaphis sacchari (Zehntner)] attack, but SbCCR2-2 was significantly induced by sorghum aphid attack. It is suggested that SbCCR2-2 is involved in the process of pest defense. Absolute quantitative real-time PCR revealed that the three CCR genes were mainly expressed in lignin deposition organs. The gene copy number of SbCCR1 was significantly higher than those of SbCCR2-1 and SbCCR2-2 in the tested tissues, especially in stem. The results provide new insight into the functions of the three CCR genes in sorghum.

Metazoan replication-dependent histone pre-mRNAs are cleaved at the 3 ′ ′ ′ ′ ′ end by U7 snRNP, an RNA-guided endonuclease that contains U7 snRNA, seven proteins of the Sm ring, FLASH, and four polyadenylation factors: symplekin, CPSF73, CPSF100, and CstF64. A fully recombinant U7 snRNP was recently reconstituted from all 13 components for functional and structural studies and shown to accurately cleave histone pre-mRNAs. Here, we analyzed the activity of recombinant U7 snRNP in more detail. We demonstrate that in addition to cleaving histone pre-mRNAs endonucleolytically, reconstituted U7 snRNP acts as a 5 ′ ′ ′ ′ ′ -3 ′ ′ ′ ′ ′ exonuclease that degrades the downstream product generated from histone pre-mRNAs as a result of the endonucleolytic cleavage. Surprisingly, recombinant U7 snRNP also acts as an endonuclease on single-stranded DNA substrates. All these activities depend on the ability of U7 snRNA to base-pair with the substrate and on the presence of the amino-terminal domain (NTD) of symplekin in either cis or trans, and are abolished by mutations within the catalytic center of CPSF73, or by binding of the NTD to the SSU72 phosphatase of RNA polymerase II. Altogether, our results demonstrate that recombinant U7 snRNP functionally mimics its endogenous counterpart and provide evidence that CPSF73 is both an endonuclease and a 5 ′ ′ ′ ′ ′ -3 ′ ′ ′ ′ ′ exonuclease, consistent with the activity of other members of the β-CASP family. Our results also raise the intriguing possibility that CPSF73 may be involved in some aspects of DNA metabolism in vivo.

The Histone Locus Body (HLB) is an evolutionarily conserved nuclear body that regulates the transcription and processing of replication-dependent (RD) histone mRNAs, which are the only eukaryotic mRNAs lacking a poly-A tail. Many nuclear bodies contain distinct domains, but how internal organization is related to nuclear body function is not fully understood. Here, we demonstrate using structured illumination microscopy that Drosophila HLBs have a “core-shell” organization in which the internal core contains transcriptionally active RD histone genes. The N-terminus of Mxc, which contains a domain required for Mxc oligomerization, HLB assembly, and RD histone gene expression, is enriched in the HLB core. In contrast, the C-terminus of Mxc is enriched in the HLB outer shell as is FLASH, a component of the active U7 snRNP that co-transcriptionally cleaves RD histone pre-mRNA. Consistent with these results, we show biochemically that FLASH binds directly to the Mxc C-terminal region. In the rapid S-M nuclear cycles of syncytial blastoderm Drosophila embryos, the HLB disassembles at mitosis and reassembles the core-shell arrangement as histone gene transcription is activated immediately after mitosis. Thus, the core-shell organization is coupled to zygotic histone gene transcription, revealing a link between HLB internal organization and RD histone gene expression.

Sudangrass, Sorghum sudanense, is an important forage in warm regions. But little is known about its genome. In this study, the transcriptomes of sudangrass S722 and sorghum Tx623B were sequenced by Illumina sequencing. More than 4Gb bases were sequenced for each library. For Tx623B and S722, 88.79% and 83.88% reads, respectively were matched to the Sorghum bicolor genome. A total of 2,397 differentially expressed genes (DEGs) were detected by RNA-Seq between the two libraries, including 849 up-regulated genes and 1,548 down-regulated genes. These DEGs could be divided into three groups by annotation analysis. A total of 44,495 single nucleotide polymorphisms (SNPs) were discovered by aligning S722 reads to the sorghum reference genome. Of these SNPs, 61.37% were transition, and this value did not differ much between different chromosomes. In addition, 16,928 insertion and deletion (indel) loci were identified between the two genomes. A total of 5,344 indel markers were designed, 15 of which were selected to construct the genetic map derived from the cross of Tx623A and Sa. It was indicated that the indel markers were useful and versatile between sorghum and sudangrass. Comparison of synonymous base substitutions (Ks) and non-synonymous base substitutions (Ka) between the two libraries showed that 95% orthologous pairs exhibited Ka/Ks<1.0, indicating that these genes were influenced by purifying selection. The results from this study provide important information for molecular genetic research and a rich resource for marker development in sudangrass and other Sorghum species.