U7 snRNP particles bind H4 PRE-mRNA in vitro

1991 ◽  
Vol 23 (10) ◽  
pp. 1049-1052 ◽  
Author(s):  
G. Pironcheva ◽  
G. Russev
Keyword(s):  
U7 Snrnp

scholarly journals Studies of the 5′ Exonuclease and Endonuclease Activities of CPSF-73 in Histone Pre-mRNA Processing

2008 ◽  
Vol 29 (1) ◽  
pp. 31-42 ◽  
Author(s):  
Xiao-cui Yang ◽  
Kelly D. Sullivan ◽  
William F. Marzluff ◽  
Zbigniew Dominski
Keyword(s):  
Cleavage Site ◽  
Complete Removal ◽  
Cleavage Product ◽  
Endonucleolytic Cleavage ◽  
Subsequent Degradation ◽  
U7 Snrnp ◽  
Space Requirements ◽  
Polyadenylation Factor

ABSTRACT Processing of histone pre-mRNA requires a single 3′ endonucleolytic cleavage guided by the U7 snRNP that binds downstream of the cleavage site. Following cleavage, the downstream cleavage product (DCP) is rapidly degraded in vitro by a nuclease that also depends on the U7 snRNP. Our previous studies demonstrated that the endonucleolytic cleavage is catalyzed by the cleavage/polyadenylation factor CPSF-73. Here, by using RNA substrates with different nucleotide modifications, we characterize the activity that degrades the DCP. We show that the degradation is blocked by a 2′-O-methyl nucleotide and occurs in the 5′-to-3′ direction. The U7-dependent 5′ exonuclease activity is processive and continues degrading the DCP substrate even after complete removal of the U7-binding site. Thus, U7 snRNP is required only to initiate the degradation. UV cross-linking studies demonstrate that the DCP and its 5′-truncated version specifically interact with CPSF-73, strongly suggesting that in vitro, the same protein is responsible for the endonucleolytic cleavage of histone pre-mRNA and the subsequent degradation of the DCP. By using various RNA substrates, we define important space requirements upstream and downstream of the cleavage site that dictate whether CPSF-73 functions as an endonuclease or a 5′ exonuclease. RNA interference experiments with HeLa cells indicate that degradation of the DCP does not depend on the Xrn2 5′ exonuclease, suggesting that CPSF-73 degrades the DCP both in vitro and in vivo.

scholarly journals Composition and processing activity of a semi-recombinant holo U7 snRNP

2019 ◽  
Vol 48 (3) ◽  
pp. 1508-1530 ◽  
Author(s):  
Katarzyna Bucholc ◽  
Wei Shen Aik ◽  
Xiao-cui Yang ◽  
Kaituo Wang ◽  
Z Hong Zhou ◽  
...  
Keyword(s):  
Animal Cells ◽  
Sm Proteins ◽  
Nuclear Extracts ◽  
In Vitro Processing ◽  
Functionally Impaired ◽  
Catalytically Active ◽  
U7 Snrnp ◽  
Core Components ◽  
Processing Activity

Abstract In animal cells, replication-dependent histone pre-mRNAs are cleaved at the 3′ end by U7 snRNP consisting of two core components: a ∼60-nucleotide U7 snRNA and a ring of seven proteins, with Lsm10 and Lsm11 replacing the spliceosomal SmD1 and SmD2. Lsm11 interacts with FLASH and together they recruit the endonuclease CPSF73 and other polyadenylation factors, forming catalytically active holo U7 snRNP. Here, we assembled core U7 snRNP bound to FLASH from recombinant components and analyzed its appearance by electron microscopy and ability to support histone pre-mRNA processing in the presence of polyadenylation factors from nuclear extracts. We demonstrate that semi-recombinant holo U7 snRNP reconstituted in this manner has the same composition and functional properties as endogenous U7 snRNP, and accurately cleaves histone pre-mRNAs in a reconstituted in vitro processing reaction. We also demonstrate that the U7-specific Sm ring assembles efficiently in vitro on a spliceosomal Sm site but the engineered U7 snRNP is functionally impaired. This approach offers a unique opportunity to study the importance of various regions in the Sm proteins and U7 snRNA in 3′ end processing of histone pre-mRNAs.

scholarly journals Cotranscriptional Processing of Drosophila Histone mRNAs

2003 ◽  
Vol 23 (12) ◽  
pp. 4046-4055 ◽  
Author(s):  
Todd E. Adamson ◽  
David H. Price
Keyword(s):  
Transcription Initiation ◽  
Divalent Cations ◽  
Histone H4 ◽  
Stem Loop ◽  
Histone Mrnas ◽  
Stem Loop Structure ◽  
U7 Snrnp ◽  
Processing Site ◽  
The One

ABSTRACT The 3′ ends of metazoan histone mRNAs are generated by specialized processing machinery that cleaves downstream of a conserved stem-loop structure. To examine how this reaction might be influenced by transcription, we used a Drosophila melanogaster in vitro system that supports both processes. In this system the complete synthesis of histone mRNA, including transcription initiation and elongation, followed by 3′ end formation, occurred at a physiologically significant rate. Processing of free transcripts was efficient and occurred with a t 1/2 of less than 1 min. Divalent cations were not required, but nucleoside triphosphates (NTPs) stimulated the rate of cleavage slightly. Isolated elongation complexes encountered a strong arrest site downstream of the mature histone H4 3′ end. In the presence of NTPs, transcripts in these arrested complexes were processed at a rate similar to that of free RNA. Removal of NTPs dramatically reduced this rate, potentially due to concealment of the U7 snRNP binding element. The arrest site was found to be a conserved feature located 32 to 35 nucleotides downstream of the processing site on the H4, H2b, and H3 genes. The significance of the newly discovered arrest sites to our understanding of the coupling between transcription and RNA processing on the one hand and histone gene expression on the other is discussed.

scholarly journals 2'-O-methyl, 2'-O-ethyl oligoribonucleotides and phosphorothioate oligodeoxyribonucleotides as inhibitors of the in vitro U7 snRNP-dependent mRNA processing event

1991 ◽  
Vol 19 (10) ◽  
pp. 2629-2635 ◽  
Author(s):  
M. Cotten ◽  
B. Oberhauser ◽  
H. Brunar ◽  
A. Holzner ◽  
G. Issakides ◽  
...  
Keyword(s):  
Mrna Processing ◽  
U7 Snrnp ◽  
Processing Event

Ultrastructural Investigations of the Contractile Filaments of Amoebae

1974 ◽  
Vol 32 ◽  
pp. 188-189
Author(s):  
P.L. Moore
Keyword(s):  
Cytoplasmic Streaming ◽  
Three Dimensional ◽  
Freeze Fracture ◽  
Amoeboid Movement ◽  
Different Types ◽  
Chemical Conditions ◽  
Complete Interpretation

Previous freeze fracture results on the intact giant, amoeba Chaos carolinensis indicated the presence of a fibrillar arrangement of filaments within the cytoplasm. A complete interpretation of the three dimensional ultrastructure of these structures, and their possible role in amoeboid movement was not possible, since comparable results could not be obtained with conventional fixation of intact amoebae. Progress in interpreting the freeze fracture images of amoebae required a more thorough understanding of the different types of filaments present in amoebae, and of the ways in which they could be organized while remaining functional.The recent development of a calcium sensitive, demembranated, amoeboid model of Chaos carolinensis has made it possible to achieve a better understanding of such functional arrangements of amoeboid filaments. In these models the motility of demembranated cytoplasm can be controlled in vitro, and the chemical conditions necessary for contractility, and cytoplasmic streaming can be investigated. It is clear from these studies that “fibrils” exist in amoeboid models, and that they are capable of contracting along their length under conditions similar to those which cause contraction in vertebrate muscles.

In Vitro Induction of Crystals of MT Protein by Vinblastine-Colcemid in Mouse Spermatids

1974 ◽  
Vol 32 ◽  
pp. 132-133
Author(s):  
John J. Wolosewick ◽  
John H. D. Bryan
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Endoplasmic Reticulum ◽  
Nuclear Ring ◽  
Rough Er ◽  
Distal Direction ◽  
In Vitro Induction

Early in spermiogenesis the manchette is rapidly assembled in a distal direction from the nuclear-ring-densities. The association of vesicles of smooth endoplasmic reticulum (SER) and the manchette microtubules (MTS) has been reported. In the mouse, osmophilic densities at the distal ends of the manchette are the organizing centers (MTOCS), and are associated with the SER. Rapid MT assembly and the lack of rough ER suggests that there is an existing pool of MT protein. Colcemid potentiates the reaction of vinblastine with tubulin and was used in this investigation to detect this protein.

Induction and Differentiation of Dental Epithelium in Vivo and in Vitro

1982 ◽  
Vol 40 ◽  
pp. 142-145
Author(s):  
E. J. Kollar
Keyword(s):  
Connective Tissue ◽  
Oral Mucosa ◽  
Basal Lamina ◽  
Functional Derivatives ◽  
Specific Source ◽  
Dental Epithelium ◽  
Connective Tissue Stroma

The differentiation and maintenance of many specialized epithelial structures are dependent on the underlying connective tissue stroma and on an intact basal lamina. These requirements are especially stringent in the development and maintenance of the skin and oral mucosa. The keratinization patterns of thin or thick cornified layers as well as the appearance of specialized functional derivatives such as hair and teeth can be correlated with the specific source of stroma which supports these differentiated expressions.

Immunoenzymatic demonstration of the simultaneous presence of transferrin, hemopexin and albumin in a same hepatocyte and their ultrastructural localization

1978 ◽  
Vol 36 (2) ◽  
pp. 88-89
Author(s):  
M. Kraemer ◽  
J. Foucrier ◽  
J. Vassy ◽  
M.T. Chalumeau
Keyword(s):  
Plasma Proteins ◽  
Rat Hepatocytes ◽  
Ultrastructural Localization ◽  
Carrier Proteins ◽  
Normal Cells ◽  
Adult Rat ◽  
Adult Rat Hepatocytes ◽  
Monospecific Antisera ◽  
Different Levels

Some authors using immunofluorescent techniques had already suggested that some hepatocytes are able to synthetize several plasma proteins. In vitro studies on normal cells or on cells issued of murine hepatomas raise the same conclusion. These works could be indications of an hepatocyte functionnal non-specialization, meanwhile the authors never give direct topographic proofs suitable with this hypothesis.The use of immunoenzymatic techniques after obtention of monospecific antisera had seemed to us useful to bring forward a better knowledge of this problem. We have studied three carrier proteins (transferrin = Tf, hemopexin = Hx, albumin = Alb) operating at different levels in iron metabolism by demonstrating and localizing the adult rat hepatocytes involved in their synthesis.Immunological, histological and ultrastructural methods have been described in a previous work.

Sem Studies of Co-Cr-Mo Surgical Implant Alloy Corrosion Behavior

1982 ◽  
Vol 40 ◽  
pp. 520-521
Author(s):  
Ann Chidester Van Orden ◽  
John L. Chidester ◽  
Anna C. Fraker ◽  
Pei Sung
Keyword(s):  
Electron Microscopy ◽  
Corrosion Behavior ◽  
Anodic Polarization ◽  
Saline Solution ◽  
Saturated Calomel Electrode ◽  
Physiological Saline ◽  
X Ray ◽  
Physiological Saline Solution ◽  
Scanning Electron

The influence of small variations in the composition on the corrosion behavior of Co-Cr-Mo alloys has been studied using scanning electron microscopy (SEM), energy dispersive x-ray analysis (EDX), and electrochemical measurements. SEM and EDX data were correlated with data from in vitro corrosion measurements involving repassivation and also potentiostatic anodic polarization measurements. Specimens studied included the four alloys shown in Table 1. Corrosion tests were conducted in Hanks' physiological saline solution which has a pH of 7.4 and was held at a temperature of 37°C. Specimens were mechanically polished to a surface finish with 0.05 µm A1203, then exposed to the solution and anodically polarized at a rate of 0.006 v/min. All voltages were measured vs. the saturated calomel electrode (s.c.e.).. Specimens had breakdown potentials near 0.47V vs. s.c.e.

Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

1982 ◽  
Vol 40 ◽  
pp. 210-211
Author(s):  
M.J. Murphy ◽  
R.R. Price ◽  
J.C. Sloman
Keyword(s):  
Cultured Cells ◽  
Human Tumor ◽  
Cell Type ◽  
Tumor Mass ◽  
Initial Cell ◽  
Cloning Assay ◽  
Human Tumor Cloning ◽  
The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

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