Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling

BACKGROUND: The polyadenylation of mRNA is one of the critical processing steps during expression of almost all eukaryotic genes. It is tightly integrated with transcription, particularly its termination, as well as other RNA processing events, i.e. capping and splicing. The poly(A) tail protects the mRNA from unregulated degradation, and it is required for nuclear export and translation initiation. In recent years, it has been demonstrated that the polyadenylation process is also involved in the regulation of gene expression. The polyadenylation process requires two components, the cis-elements on the mRNA and a group of protein factors that recognize the cis-elements and produce the poly(A) tail. Here we report a comprehensive pairwise protein-protein interaction mapping and gene expression profiling of the mRNA polyadenylation protein machinery in Arabidopsis. RESULTS: By protein sequence homology search using human and yeast polyadenylation factors, we identified 28 proteins that may be components of Arabidopsis polyadenylation machinery. To elucidate the protein network and their functions, we first tested their protein-protein interaction profiles. Out of 320 pair-wise protein-protein interaction assays done using the yeast two-hybrid system, 56 (approximately 17%) showed positive interactions. 15 of these interactions were further tested, and all were confirmed by co-immunoprecipitation and/or in vitro co-purification. These interactions organize into three distinct hubs involving the Arabidopsis polyadenylation factors. These hubs are centered around AtCPSF100, AtCLPS, and AtFIPS. The first two are similar to complexes seen in mammals, while the third one stands out as unique to plants. When comparing the gene expression profiles extracted from publicly available microarray datasets, some of the polyadenylation related genes showed tissue-specific expression, suggestive of potential different polyadenylation complex configurations. CONCLUSION: An extensive protein network was revealed for plant polyadenylation machinery, in which all predicted proteins were found to be connecting to the complex. The gene expression profiles are indicative that specialized sub-complexes may be formed to carry out targeted processing of mRNA in different developmental stages and tissue types. These results offer a roadmap for further functional characterizations of the protein factors, and for building models when testing the genetic contributions of these genes in plant growth and development

A genetic variation map for chicken with 2.8 million single-nucleotide polymorphisms

We describe a genetic variation map for the chicken genome containing 2.8 million single-nucleotide polymorphisms ( SNPs). This map is based on a comparison of the sequences of three domestic chicken breeds ( a broiler, a layer and a Chinese silkie) with that of their wild ancestor, red jungle fowl. Subsequent experiments indicate that at least 90% of the variant sites are true SNPs, and at least 70% are common SNPs that segregate in many domestic breeds. Mean nucleotide diversity is about five SNPs per kilobase for almost every possible comparison between red jungle fowl and domestic lines, between two different domestic lines, and within domestic lines - in contrast to the notion that domestic animals are highly inbred relative to their wild ancestors. In fact, most of the SNPs originated before domestication, and there is little evidence of selective sweeps for adaptive alleles on length scales greater than 100 kilobases

Heritable Targeted Inactivation of Myostatin Gene in Yellow Catfish (Pelteobagrus fulvidraco) Using Engineered Zinc Finger Nucleases

Yellow catfish (Pelteobagrus fulvidraco) is one of the most important freshwater aquaculture species in China. However, its small size and lower meat yield limit its edible value. Myostatin (MSTN) is a negative regulator of mammalian muscle growth. But, the function of Mstn in fish remains elusive. To explore roles of mstn gene in fish growth and create a strain of yellow catfish with high amount of muscle mass, we performed targeted disruption of mstn in yellow catfish using engineered zinc-finger nucleases (ZFNs). Employing zebrafish embryos as a screening system to identify ZFN activity, we obtained one pair of ZFNs that can edit mstn in yellow catfish genome. Using the ZFNs, we successfully obtained two founders (Founder July29-7 and Founder July29-8) carrying mutated mstn gene in their germ cells. The mutated mstn allele inherited from Founder July29-7 was a null allele (mstnnju6) containing a 4 bp insertion, predicted to encode function null Mstn. The mutated mstn inherited from Founder July29-8 was a complex type of mutation (mstnnju7), predicted to encode a protein lacking two amino acids in the N-terminal secretory signal of Mstn. Totally, we obtained 6 mstnnju6/+ and 14 mstnnju7/+ yellow catfish. To our best knowledge, this is the first endogenous gene knockout in aquaculture fish. Our result will help in understanding the roles of mstn gene in fish

Identification and characterization of zebrafish fibronectins

The goal of this study was to identify the fibronectin (FN) isoforms present in the zebrafish and determine their structure and pattern of expression. Sequence analysis of zebrafish FN cDNAs indicates that at least two forms of the protein exist in fish. One form (FN1) is very similar to FNs identified in other vertebrates possessing 12 type I, 2 type II, 17 type III repeats including two alternative splice sites (EIIIA and EIIIB) and a variable region (V). It exhibits 59% identity with Xenopus FN. Partial sequence analysis of zebrafish FN genomic DNA reveals that the FN gene organization in zebrafish is also similar to other vertebrates. However, unlike other vertebrates, zebrafish FN1 does not produce any splicing variant at the EIIIB, EIIIA and V regions. FN1 mRNA is widely present in zebrafish embryos and adult tissues. Whole mount in situ hybridization shows FNl mRNA is stored at early cleavage and blastula stages and present in the regions that are involved in cell movement during gastrulation. Unlike mouse and chicken, zebrafish does not possess obvious level of FN1 message in the regions of the developing somites and notochord during segmentation, but FN1 mRNA is abundant in otic vesicle during early ear development. In addition to FN1, zebrafish has a novel truncated form of FN (FN2). The predicted structure of FN2 is identical to FN1 at the N-terminal region possessing 9 type I, 2 type II and the first three type III repeats. Following the third type III repeat, the protein contains a unique 20 amino acid C-terminal tail that is completely different from the C-terminus of FN. Sequence analysis of FN genomic DNA demonstrates that FN2 is a unique splice variant of FN1. Like FN1, FN2 message is also widely present in zebrafish embryos and adult tissues although its abundance is much lower than FN1. The production of zebrafish FNs was investigated in a zebrafish liver cell line (ZFL). Results show that ZFL cells secrete FN proteins that can bind fish gelatin but not pork gelatin. The expression of the proteins in ZFL cells is not regulated by TGFP and retinoic acid

Excessive nitrite affects zebrafish valvulogenesis through yielding too much NO signaling.

Sodium nitrite, a common food additive, exists widely not only in the environment but also in our body. Excessive nitrite causes toxicological effects on human health; however, whether it affects vertebrate heart valve development remains unknown. In vertebrates, developmental defects of cardiac valves usually lead to congenital heart disease. To understand the toxic effects of nitrite on valvulogenesis, we exposed zebrafish embryos with different concentrations of sodium nitrite. Our results showed that sodium nitrite caused developmental defects of zebrafish heart dose dependently. It affected zebrafish heart development starting from 36 hpf (hour post fertilization) when heart initiates looping process. Comprehensive analysis on the embryos at 24 hpf and 48 hpf showed that excessive nitrite did not affect blood circulation, vascular network, myocardium and endocardium development. But development of endocardial cells in atrioventricular canal (AVC) of the embryos at 48 hpf was disrupted by too much nitrite, leading to defective formation of primitive valve leaflets at 76 hpf. Consistently, excessive nitrite diminished expressions of valve progenitor markers including bmp4, has2, vcana and notch1b at 48 hpf. Furthermore, 3', 5'-cyclic guanosine monophosphate (cGMP), downstream of nitric oxide (NO) signaling, was increased its level significantly in the embryos exposed with excessive nitrite and microinjection of soluble guanylate cyclase inhibitor ODQ (1H-[1], [2], [4]Oxadiazolo[4,3-a] quinoxalin-1-one), an antagonist of NO signaling, into nitrite-exposed embryos could partly rescue the cardiac valve malformation. Taken together, our results show that excessive nitrite affects early valve leaflet formation by producing too much NO signaling

A Novel Plant in Vitro Assay System for Pre-mRNA Cleavage during 3′-End Formation1[OA]

Messenger RNA (mRNA) maturation in eukaryotic cells requires the formation of the 3′ end, which includes two tightly coupled steps: the committing cleavage reaction that requires both correct cis-element signals and cleavage complex formation, and the polyadenylation step that adds a polyadenosine [poly(A)] tract to the newly generated 3′ end. An in vitro biochemical assay plays a critical role in studying this process. The lack of such an assay system in plants hampered the study of plant mRNA 3′-end formation for the last two decades. To address this, we have now established and characterized a plant in vitro cleavage assay system, in which nuclear protein extracts from Arabidopsis (Arabidopsis thaliana) suspension cell cultures can accurately cleave different pre-mRNAs at expected in vivo authenticated poly(A) sites. The specific activity is dependent on appropriate cis-elements on the substrate RNA. When complemented by yeast (Saccharomyces cerevisiae) poly(A) polymerase, about 150-nucleotide poly(A) tracts were added specifically to the newly cleaved 3′ ends in a cooperative manner. The reconstituted polyadenylation reaction is indicative that authentic cleavage products were generated. Our results not only provide a novel plant pre-mRNA cleavage assay system, but also suggest a cross-kingdom functional complementation of yeast poly(A) polymerase in a plant system

A Toolpath Planning Method for Optical Freeform Surface Ultra-Precision Turning Based on NURBS Surface Curvature

As the applications for freeform optical surfaces continue to grow, the need for high-precision machining methods is becoming more and more of a necessity. Different toolpath strategies for the ultra-high precision turning of freeform surfaces can have a significant impact on the quality of the machined surfaces. This paper presents a novel toolpath planning method for ultra-precision slow tool servo diamond turning based on the curvature of freeform surfaces. The method analyzes the differential geometric properties of freeform surfaces by reconstructing NURBS freeform surfaces. A mathematical model is constructed based on the parameters of different positions of the freeform surface, toolpath parameters, and tool residual height. Appropriate toolpath parameters can be calculated to generate the optical freeform ultra-precision slow tool servo diamond turning toolpath. Compared with the toolpaths generated by the traditional Archimedes spiral method, the ultra-precision slow tool servo diamond turning toolpath planning method proposed in this paper can generate more uniform toolpaths on the freeform surfaces and keep the residual tool height within a small range

Sodium nitrite caused defective development of zebrafish heart in a dose dependent way.

<p>(A–C) Zebrafish embryos exposed to 100 mg/l sodium nitrite from 10 hpf exhibited cardiac edema from slight phenotype (B) to severe phenotype (C) compared to control embryos with normal heart (A) at 108 hpf. (D) A scatter plot showing the edema index (EI) of embryos exposed with different concentration of sodium nitrite. EI is defined as b/a. Average ± standard errors of EIs were shown in lines. (E–G) Histological sections showing 100 mg/l sodium nitrite exposure caused defective structure of zebrafish heart at 108 hpf. Compared to control embryos with normal pericardial membrane, myocardium and both superior and inferior valve leaflets (E), 4/7 embryos exposed to the nitrite showed thinner pericardial membrane and myocardium, and only superior valve leaflet but no formation of inferior valve leaflet (F), whereas 3/7 of the treated embryos displayed thinner pericardial membrane and myocardium and no formation of either superior or inferior leaflets (G). a: the semi-diameter of ventricle; b: the semi-diameter of the pericardial cavity. Red star (*): pericardial membrane; Black star (*): myocardium; Black arrow: position of superior valve leaflet; Black arrowhead: position of inferior valve leaflet; A: atria; V: ventricle.</p