Lingdi Zhang

Structural evidence for consecutive Hel308-like modules in the spliceosomal ATPase Brr2.

Brr2 is a DExD/H-box helicase responsible for U4/U6 unwinding during spliceosomal activation. Brr2 contains two helicase-like domains, each of which is followed by a Sec63 domain with unknown function. We determined the crystal structure of the second Sec63 domain, which unexpectedly resembles domains 4 and 5 of DNA helicase Hel308. This, together with sequence similarities between Brr2s helicase-like domains and domains 1–3 of Hel308, led us to hypothesize that Brr2 contains two consecutive Hel308-like modules (Hel308-I and Hel308-II). Our structural model and mutagenesis data suggest that Brr2 shares a similar helicase mechanism with Hel308. We demonstrate that Hel308-II interacts with Prp8 and Snu114 in vitro and in vivo. We further find that the C-terminal region of Prp8 (Prp8-CTR) facilitates the binding of the Brr2–Prp8-CTR complex to U4/U6. Our results have important implications for the mechanism and regulation of Brr2s activity in splicing.

Comprehensive in vivo RNA-binding site analyses reveal a role of Prp8 in spliceosomal assembly

Prp8 stands out among hundreds of splicing factors as a protein that is intimately involved in spliceosomal activation and the catalytic reaction. Here, we present the first comprehensive in vivo RNA footprints for Prp8 in budding yeast obtained using CLIP (cross-linking and immunoprecipitation)/CRAC (cross-linking and analyses of cDNAs) and next-generation DNA sequencing. These footprints encompass known direct Prp8-binding sites on U5, U6 snRNA and intron-containing pre-mRNAs identified using site-directed cross-linking with in vitro assembled small nuclear ribonucleoproteins (snRNPs) or spliceosome. Furthermore, our results revealed novel Prp8-binding sites on U1 and U2 snRNAs. We demonstrate that Prp8 directly cross-links with U2, U5 and U6 snRNAs and pre-mRNA in purified activated spliceosomes, placing Prp8 in position to bring the components of the active site together. In addition, disruption of the Prp8 and U1 snRNA interaction reduces tri-snRNP level in the spliceosome, suggesting a previously unknown role of Prp8 in spliceosomal assembly through its interaction with U1 snRNA.

On the removal of material along a polishing path by fixed abrasives

Abstract Material is removed when a surface is polished. This paper presents a model of the removal of material along a polishing path when a surface is polished by fixed abrasives. The proposed model assumes that the pressure distribution is Hertzian at the contact between the tool and the surface and the abrading rate follows the Archard wear law. According to the model, the profile of material removal is parabolic, and the depth and width of the profile may depend on the tool orientation. Polishing experiments are presented for the validation of the proposed model. Examples are also given to illustrate the applications of the model.

Brr2 plays a role in spliceosomal activation in addition to U4/U6 unwinding

Brr2 is a DExD/H-box RNA helicase that is responsible for U4/U6 unwinding, a critical step in spliceosomal activation. Brr2 is a large protein (∼250 kD) that consists of an N-terminal domain (∼500 residues) with unknown function and two Hel308-like modules that are responsible for RNA unwinding. Here we demonstrate that removal of the entire N-terminal domain is lethal to Saccharomyces cerevisiae and deletion of the N-terminal 120 residues leads to splicing defects and severely impaired growth. This N-terminal truncation does not significantly affect Brr2s helicase activity. Brr2-Δ120 can be successfully assembled into the tri-snRNP (albeit at a lower level than the WT Brr2) and the spliceosomal B complex. However, the truncation significantly impairs spliceosomal activation, leading to a dramatic reduction of U5, U6 snRNAs and accumulation of U1 snRNA in the Bact complex. The N-terminal domain of Brr2 does not seem to be directly involved in regulating U1/5ss unwinding. Instead, the N-terminal domain seems to be critical for retaining U5 and U6 snRNPs during/after spliceosomal activation through its interaction with snRNAs and possibly other spliceosomal proteins, revealing a new role of Brr2 in spliceosomal activation in addition to U4/U6 unwinding.

On cutting forces in peripheral milling of curved surfaces

Abstract This paper presents the development of an analytical model of cutting forces in peripheral milling of curved surfaces. The effect of the workpiece curvature is taken into account in the model construction. Based on the relationship of differential cutting forces and chip load, the total cutting forces are formulated by integration of the differential cutting forces along the cutting flutes in the feed and cross-feed directions. This formulation leads to an explicit expression for the force waveforms as algebraic functions of cutter variables, process parameters, machining configuration and workpiece geometry. Experiments are performed over a wide range of conditions to verify the model and meanwhile the effects of the process parameters on the cutting forces are revealed. The presented model facilitates force estimation and provides a basis for the analysis, prediction and improvement of surface accuracy in peripheral milling of curved surfaces.

CryoEM structure of Saccharomyces cerevisiae U1 snRNP offers insight into alternative splicing.

U1 snRNP plays a critical role in 5ʹ-splice site recognition and is a frequent target of alternative splicing factors. These factors transiently associate with human U1 snRNP and are not amenable for structural studies, while their Saccharomyces cerevisiae (yeast) homologs are stable components of U1 snRNP. Here, we report the cryoEM structure of yeast U1 snRNP at 3.6u2009Å resolution with atomic models for ten core proteins, nearly all essential domains of its RNA, and five stably associated auxiliary proteins. The foot-shaped yeast U1 snRNP contains a core in the “ball-and-toes” region architecturally similar to the human U1 snRNP. All auxiliary proteins are in the “arch-and-heel” region and connected to the core through the Prp42/Prp39 paralogs. Our demonstration that homodimeric human PrpF39 directly interacts with U1C-CTD, mirroring yeast Prp42/Prp39, supports yeast U1 snRNP as a model for understanding how transiently associated auxiliary proteins recruit human U1 snRNP in alternative splicing.U1 snRNP is critical for 5′ splicing site recognition in pre-mRNA splicing. Here the authors describe the cryo-EM structure of the yeast U1 snRNP and suggest that PrpF39 is an alternative splicing factor essential for the successful recruitment of U1 snRNP by other alternative splicing factors.

Structural analyses of the pre‐mRNA splicing machinery

Pre‐mRNA splicing is a critical event in the gene expression pathway of all eukaryotes. The splicing reaction is catalyzed by the spliceosome, a huge protein‐RNA complex that contains five snRNAs and hundreds of different protein factors. Understanding the structure of this large molecular machinery is critical for understanding its function. Although the highly dynamic nature of the spliceosome, in both composition and conformation, posed daunting challenges to structural studies, there has been significant recent progress on structural analyses of the splicing machinery, using electron microscopy, crystallography, and nuclear magnetic resonance. This review discusses key recent findings in the structural analyses of the spliceosome and its components and how these findings advance our understanding of the function of the splicing machinery.

miR-182 selectively targets HOXA10 in goat endometrial epithelium cells in vitro

Proper HOXA10 expression was essential for endometrial receptivity what was crucial for successful embryo implantation in mammalian. This study confirmed that miR-182 regulated the expression levels of HOXA10 by binding to its 3 UTR, selectively downregulated HOXA10 in goat endometrial epithelium cells (gEECs) but not stromal cell (gESCs) in vitro. However, HOXA10 and miR-182 both up-expressed in the goat endometrium at gestational day 15 (D15) compared with gestational day 5 (D5), suggesting that there were some other factors regulated the expression of HOXA10 during the development of goat endometrium in vivo. Whats more, HOXA10 gene silencing (HOXA10-siRNA) resulted in gEECs apoptosis in vitro, and it regulated the protein levels of oestrogen receptor a (ERa), progesterone receptor B (PRb), insulin-like growth factor 1 receptor (IGF1R), BCL-2, pleiotrophin (PTN), AKT and p-JNK in gEECs. Furthermore, HOXA10 might regulate the protein levels of endometrial receptivity biomarker genes, including vascular endothelial growth factor (VEGF), osteopontin (OPN), cyclooxygenase-2 (COX-2) and prolactin receptor (PRLR) in gEECs. In conclusion, miR-182 targeted HOXA10 selectively in EECs in vitro, and HOXA10 played an important role in maintaining the function of EECs in dairy goats.

Eya3 partners with PP2A to induce c-Myc stabilization and tumor progression

Eya genes encode a unique family of multifunctional proteins that serve as transcriptional co-activators and as haloacid dehalogenase-family Tyr phosphatases. Intriguingly, the N-terminal domain of Eyas, which does not share sequence similarity to any known phosphatases, contains a separable Ser/Thr phosphatase activity. Here, we demonstrate that the Ser/Thr phosphatase activity of Eya is not intrinsic, but arises from its direct interaction with the protein phosphatase 2A (PP2A)-B55α holoenzyme. Importantly, Eya3 alters the regulation of c-Myc by PP2A, increasing c-Myc stability by enabling PP2A-B55α to dephosphorylate pT58, in direct contrast to the previously described PP2A-B56α-mediated dephosphorylation of pS62 and c-Myc destabilization. Furthermore, Eya3 and PP2A-B55α promote metastasis in a xenograft model of breast cancer, opposing the canonical tumor suppressive function of PP2A-B56α. Our study identifies Eya3 as a regulator of PP2A,xa0a major cellular Ser/Thr phosphatase, and uncovers a mechanism of controlling the stability of a critical oncogene, c-Myc.Eya proteins are characterised by phosphatase activity associated with both the evolutionary conserved region and the less conserved N-terminal domain (NTD). Here the authors show that NTD mediates the interaction with PP2A and regulates c-Myc phosphorylation and stability, potentially switching PP2A from a tumour suppressor to an oncogene.

The Eya phosphatase: Its unique role in cancer

The Eya proteins were originally identified as essential transcriptional co-activators of the Six family of homeoproteins. Subsequently, the highly conserved C-terminal domains of the Eya proteins were discovered to act as a Mg2+-dependent Tyr phosphatases, making Eyas the first transcriptional activators to harbor intrinsic phosphatase activity. Only two direct targets of the Eya Tyr phosphatase have been identified: H2AX, whose dephosphorylation directs cells to the DNA repair instead of the apoptotic pathway upon DNA damage, and ERβ, whose dephosphorylation inhibits its anti-tumor transcriptional activity. The Eya Tyr phosphatase mediates breast cancer cell transformation, migration, invasion, as well as metastasis, through targets not yet identified. Intriguingly, the N-terminal domain of Eya contains a separate Ser/Thr phosphatase activity implicated in innate immunity and in regulating c-Myc stability. Thus, Eya proteins are highly complex, containing two separable phosphatase domains and a transcriptional activation domain, thereby influencing tumor progression through multiple mechanisms.