scholarly journals Functional organization of box C/D RNA-guided RNA methyltransferase

2020 ◽  
Vol 48 (9) ◽  
pp. 5094-5105
Author(s):  
Zuxiao Yang ◽  
Jiayin Wang ◽  
Lin Huang ◽  
David M J Lilley ◽  
Keqiong Ye
Keyword(s):  
Recombinant Proteins ◽  
Mechanism Of Action ◽  
Functional Organization ◽  
Protein Complexes ◽  
Site Specific ◽  
Guide Rnas ◽  
Chaetomium Thermophilum ◽  
Rna Protein Complexes ◽  
Insight Into

Abstract Box C/D RNA protein complexes (RNPs) catalyze site-specific 2′-O-methylation of RNA with specificity determined by guide RNAs. In eukaryotic C/D RNP, the paralogous Nop58 and Nop56 proteins specifically associate with terminal C/D and internal C'/D' motifs of guide RNAs, respectively. We have reconstituted active C/D RNPs with recombinant proteins of the thermophilic yeast Chaetomium thermophilum. Nop58 and Nop56 could not distinguish between the two C/D motifs in the reconstituted enzyme, suggesting that the assembly specificity is imposed by trans-acting factors in vivo. The two C/D motifs are functionally independent and halfmer C/D RNAs can also guide site-specific methylation. Extensive pairing between C/D RNA and substrate is inhibitory to modification for both yeast and archaeal C/D RNPs. N6-methylated adenine at box D/D' interferes with the function of the coupled guide. Our data show that all C/D RNPs share the same functional organization and mechanism of action and provide insight into the assembly specificity of eukaryotic C/D RNPs.

RNA and RNA–Protein Complex Crystallography and its Challenges

2014 ◽  
Vol 67 (12) ◽  
pp. 1741 ◽  
Author(s):  
Janine K. Flores ◽  
James L. Walshe ◽  
Sandro F. Ataide
Keyword(s):  
Protein Complex ◽  
Protein Complexes ◽  
Future Directions ◽  
Vast Number ◽  
X Ray Crystallography ◽  
Rna Biology ◽  
Non Coding Rnas ◽  
Rna Protein Complexes

RNA biology has changed completely in the past decade with the discovery of non-coding RNAs. Unfortunately, obtaining mechanistic information about these RNAs alone or in cellular complexes with proteins has been a major problem. X-ray crystallography of RNA and RNA–protein complexes has suffered from the major problems encountered in preparing and purifying them in large quantity. Here, we review the available techniques and methods in vitro and in vivo used to prepare and purify RNA and RNA–protein complex for crystallographic studies. We also discuss the future directions necessary to explore the vast number of RNA species waiting for their atomic-resolution structure to be determined.

scholarly journals THE MECHANISM OF ACTION OF ANTI-LYMPHOCYTE SERUM

1969 ◽  
Vol 130 (1) ◽  
pp. 49-76 ◽  
Author(s):  
Eugene M. Lance
Keyword(s):  
Mechanism Of Action ◽  
Strong Support ◽  
Reticuloendothelial System ◽  
Selective Ablation ◽  
Subsequent Elution ◽  
Radionuclide Tracer ◽  
Rapid Clearance ◽  
Distribution Studies ◽  
Insight Into

Studies designed to gain insight into the mechanism of action of the active component of antilymphocyte serum were carried out using an antibody eluate prepared from the IgG fraction of anti-lymphocyte serum by absorption and subsequent elution from thymocyte membrane. The resulting antibody eluate was labeled with radionuclide tracer to determine the fate of the antibody in vivo. The result indicated that anti-lymphocytic antibodies are eliminated from recipients extremely rapidly. The mechanism for this rapid clearance appears to depend upon the absorption of antibody molecules onto lymphocyte surfaces and the subsequent clearing and degradation of the antibody-lymphocyte complexes by the reticuloendothelial system. Distribution studies confirm that the major site of antibody-lymphocyte interaction occurs in the periphery with relatively little penetration of antibody within lymphoid organs. Radioautographic studies showed that the pattern of localization within lymphoid and other organs is confined to rather specific areas. These observations are believed to offer strong support for the notion that anti-lymphocyte serum achieves its immunosuppressive effect by bringing about a selective ablation of the population of recirculating lymphocytes.

scholarly journals Box C/D guide RNAs recognize a maximum of 10 nt of substrates

2016 ◽  
Vol 113 (39) ◽  
pp. 10878-10883 ◽  
Author(s):  
Zuxiao Yang ◽  
Jinzhong Lin ◽  
Keqiong Ye
Keyword(s):  
Substrate Recognition ◽  
Active Conformation ◽  
Base Pairs ◽  
Recognition Mechanism ◽  
Site Specific ◽  
Guide Rnas ◽  
Modification Activity ◽  
Guide Sequence ◽  
Protein Channel ◽  
Insight Into

Box C/D RNAs guide site-specific 2′-O-methylation of RNAs in archaea and eukaryotes. The spacer regions between boxes C to D′ and boxes C′ to D contain the guide sequence that can form a stretch of base pairs with substrate RNAs. The lengths of spacer regions and guide-substrate duplexes are variable among C/D RNAs. In a previously determined structure of C/D ribonucleoprotein (RNP), a 12-nt-long spacer forms 10 bp with the substrate. How spacers and guide–substrate duplexes of other lengths are accommodated remains unknown. Here we analyze how the lengths of spacers and guide-substrate duplexes affect the modification activity and determine three structures of C/D RNPs assembled with different spacers and substrates. We show that the guide can only form a duplex of a maximum of 10 bp with the substrate during modification. Slightly shorter duplexes are tolerated, but longer duplexes must be unwound to fit into a capped protein channel for modification. Spacers with <12 nucleotides are defective, mainly because they cannot load the substrate in the active conformation. For spacers with >12 nucleotides, the excessive unpaired sequences near the box C/C′ side are looped out. Our results provide insight into the substrate recognition mechanism of C/D RNA and refute the RNA-swapped model for dimeric C/D RNP.

scholarly journals Enzyme Complexes of Ptr4CL and PtrHCT Modulate Co-enzyme A Ligation of Hydroxycinnamic Acids for Monolignol Biosynthesis in Populus trichocarpa

2021 ◽  
Vol 12 ◽  
Author(s):  
Chien-Yuan Lin ◽  
Yi Sun ◽  
Jina Song ◽  
Hsi-Chuan Chen ◽  
Rui Shi ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Metabolic Flux ◽  
Protein Complexes ◽  
Lignin Content ◽  
Populus Trichocarpa ◽  
Wood Formation ◽  
Hydroxycinnamic Acids ◽  
Monolignol Biosynthesis ◽  
Diverse Plant Species

Co-enzyme A (CoA) ligation of hydroxycinnamic acids by 4-coumaric acid:CoA ligase (4CL) is a critical step in the biosynthesis of monolignols. Perturbation of 4CL activity significantly impacts the lignin content of diverse plant species. In Populus trichocarpa, two well-studied xylem-specific Ptr4CLs (Ptr4CL3 and Ptr4CL5) catalyze the CoA ligation of 4-coumaric acid to 4-coumaroyl-CoA and caffeic acid to caffeoyl-CoA. Subsequently, two 4-hydroxycinnamoyl-CoA:shikimic acid hydroxycinnamoyl transferases (PtrHCT1 and PtrHCT6) mediate the conversion of 4-coumaroyl-CoA to caffeoyl-CoA. Here, we show that the CoA ligation of 4-coumaric and caffeic acids is modulated by Ptr4CL/PtrHCT protein complexes. Downregulation of PtrHCTs reduced Ptr4CL activities in the stem-differentiating xylem (SDX) of transgenic P. trichocarpa. The Ptr4CL/PtrHCT interactions were then validated in vivo using biomolecular fluorescence complementation (BiFC) and protein pull-down assays in P. trichocarpa SDX extracts. Enzyme activity assays using recombinant proteins of Ptr4CL and PtrHCT showed elevated CoA ligation activity for Ptr4CL when supplemented with PtrHCT. Numerical analyses based on an evolutionary computation of the CoA ligation activity estimated the stoichiometry of the protein complex to consist of one Ptr4CL and two PtrHCTs, which was experimentally confirmed by chemical cross-linking using SDX plant protein extracts and recombinant proteins. Based on these results, we propose that Ptr4CL/PtrHCT complexes modulate the metabolic flux of CoA ligation for monolignol biosynthesis during wood formation in P. trichocarpa.

scholarly journals MBD2/NuRD and MBD3/NuRD, Two Distinct Complexes with Different Biochemical and Functional Properties

2006 ◽  
Vol 26 (3) ◽  
pp. 843-851 ◽  
Author(s):  
Xavier Le Guezennec ◽  
Michiel Vermeulen ◽  
Arie B. Brinkman ◽  
Wieteke A. M. Hoeijmakers ◽  
Adrian Cohen ◽  
...  
Keyword(s):  
Functional Properties ◽  
Chromatin Immunoprecipitation ◽  
Physiological Response ◽  
Protein Complexes ◽  
Cpg Islands ◽  
Dependent Manner ◽  
Putative Tumor Suppressor ◽  
Protein Tagging ◽  
Insight Into

ABSTRACT The human genome contains a number of methyl CpG binding proteins that translate DNA methylation into a physiological response. To gain insight into the function of MBD2 and MBD3, we first applied protein tagging and mass spectrometry. We show that MBD2 and MBD3 assemble into mutually exclusive distinct Mi-2/NuRD-like complexes, called MBD2/NuRD and MBD3/NuRD. We identified DOC-1, a putative tumor suppressor, as a novel core subunit of MBD2/NuRD as well as MBD3/NuRD. PRMT5 and its cofactor MEP50 were identified as specific MBD2/NuRD interactors. PRMT5 stably and specifically associates with and methylates the RG-rich N terminus of MBD2. Chromatin immunoprecipitation experiments revealed that PRMT5 and MBD2 are recruited to CpG islands in a methylation-dependent manner in vivo and that H4R3, a substrate of PRMT, is methylated at these loci. Our data show that MBD2/NuRD and MBD3/NuRD are distinct protein complexes with different biochemical and functional properties.

Structural basis for site-specific ribose methylation by box C/D RNA protein complexes

Nature ◽  
2011 ◽  
Vol 469 (7331) ◽  
pp. 559-563 ◽  
Author(s):  
Jinzhong Lin ◽  
Shaomei Lai ◽  
Ru Jia ◽  
Anbi Xu ◽  
Liman Zhang ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Structural Basis ◽  
Site Specific ◽  
Rna Protein Complexes

scholarly journals The L7Ae proteins mediate a widespread and highly functional protein–RNA interaction

2019 ◽  
Vol 41 (2) ◽  
pp. 40-44
Author(s):  
David M.J. Lilley
Keyword(s):  
Protein Complexes ◽  
Structural Motif ◽  
Rna Modification ◽  
Functional Protein ◽  
Site Specific ◽  
Rna Interaction ◽  
Rna Protein Complexes

The k-turn is a ubiquitous structural motif in RNA forming a very tight kink in the axis of helical RNA that plays an important role in many aspects of RNA function. L7Ae is a member of a superfamily of proteins that bind k-turns in RNA, stabilizing the tightly kinked conformation. They are extremely widespread and are important in the assembly of RNA–protein complexes central to translation, splicing and site-specific RNA modification. The interaction is exploited in order to regulate the synthesis of L7Ae proteins and is itself subject to regulation in box C/D snoRNP assembly by N6 methylation of a key adenine in the k-turn. Lastly, we can exploit the L7Ae–k-turn interaction in the construction of nanoscale assemblies.

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