scholarly journals Fast and unbiased purification of RNA-protein complexes after UV cross-linking

2019 ◽  
Author(s):  
Erika C Urdaneta ◽  
Benedikt M Beckmann
Keyword(s):  
Protein Interactions ◽  
Protein Complexes ◽  
Regulation Of Gene Expression ◽  
Source Material ◽  
Regulatory Rna ◽  
Polyadenylated Rna ◽  
Simple Protocol ◽  
The Individual ◽  
Post Transcriptional Regulation ◽  
Rna Protein Complexes

Post-transcriptional regulation of gene expression in cells is facilitated by formation of RNA-protein complexes (RNPs). While many methods to study eukaryotic (m)RNPs rely on purification of polyadenylated RNA, other important regulatory RNA classes or bacterial mRNA could not be investigated at the same depth. To overcome this limitation, we developed Phenol Toluol extraction (PTex), a novel and unbiased method for the purification of UV cross-linked RNPs in living cells. PTex is a fast (2-3 hrs) and simple protocol. The purification principle is solely based on physicochemical properties of cross-linked RNPs, enabling us to interrogate RNA-protein interactions system-wide and beyond poly(A) RNA from a variety of species and source material. Here, we are presenting an introduction of the underlying separation principles and give a detailed discussion of the individual steps as well as incorporation of PTex in high-throughput pipelines.

scholarly journals Characterization of major chromatin assembly proteins in tetrahymena thermophile using proeomic and molecular evolutionary analyses

2021 ◽  
Author(s):  
Syed N Shah
Keyword(s):  
Protein Interactions ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Regulation Of Gene Expression ◽  
Chromatin Assembly ◽  
Histone Chaperones ◽  
Protein Protein Interactions ◽  
Selective Forces ◽  
Assembly Proteins

Histones H3/H4 are deposited onto DNA in a replication-dependent or independent fashion by the CAF1 and HIRA protein complexes. Despite the identification of these protein complexes, mechanistic details remain unclear. Recently, we showed that in T. thermophila histone chaperones Nrp1, Asf1 and the Impβ6 importin function together to transport newly synthesized H3/H4 from the cytoplasm to the nucleus. To characterize chromatin assembly proteins in T.thermophila, I used affinity purification combined with mass spectrometry to identify protein-protein interactions of Nrp1, Cac2 subunit of CAF1, HIRA and histone modifying Hat1-complex in T. thermophila. I found that the three-subunit T.thermophila CAF1 complex interacts with Casein Kinase 2 (CKII), possibly accounting for previously reported human CAF1phosphorylation. I also found that Hat2 subunit of HAT1 complex is also shared by CAF1 complex as its Cac3 subunit. This suggests that Hat2/Cac3 might exist in two separate pools of protein complexes. Remarkably, proteomic analysis of Hat2/Cac3 in turn revealed that it forms several complexes with other proteins including SIN3, RXT3, LIN9 and TESMIN, all of which have known roles in the regulation of gene expression. Finally, I asked how selective forces might have impacted on the function of proteins involved in H3/H4 transport. Focusing on NASP which possesses several TPR motifs, I showed that its protein-protein interactions are conserved in T. thermophila. Using molecular evolutionary methods I show that different TPRs in NASP evolve at different rates possibly accounting for the functional diversity observed among different family members.

scholarly journals Chromatin-associated protein complexes link DNA base J and transcription termination in Leishmania

2020 ◽  
Author(s):  
Bryan C Jensen ◽  
Isabelle Q. Phan ◽  
Jacquelyn R. McDonald ◽  
Aakash Sur ◽  
Mark A. Gillespie ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Protein Complexes ◽  
Transcription Termination ◽  
Critical Role ◽  
Regulation Of Gene Expression ◽  
Regulation Of Transcription ◽  
Control Of Gene Expression ◽  
Polycistronic Transcription ◽  
Post Transcriptional Regulation

AbstractUnlike most other eukaryotes, Leishmania and other trypanosomatid protozoa have largely eschewed transcriptional control of gene expression; relying instead on post-transcriptional regulation of mRNAs derived from polycistronic transcription units (PTUs). In these parasites, a novel modified nucleotide base (β-D-glucopyranosyloxymethyluracil) known as J plays a critical role in ensuring that transcription termination occurs only at the end of each PTU, rather than at the polyadenylation sites of individual genes. To further understand the biology of J-associated processes, we used tandem affinity purification (TAP-tagging) and mass spectrometry to reveal proteins that interact with the glucosyltransferase performing the final step in J synthesis. These studies identified four proteins reminiscent of subunits in the PTW/PP1 complex that controls transcription termination in higher eukaryotes. Moreover, bioinformatic analyses identified the DNA-binding subunit of Leishmania PTW/PP1 as a novel J-binding protein (JBP3). Down-regulation of JBP3 expression levels in Leishmania resulted in a substantial increase in transcriptional read-through at the 3’ end of most PTUs. Additional TAP-tagging experiments showed that JBP3 also associates with two other protein complexes. One consists of subunits with domains suggestive of a role in chromatin modification/remodeling; while the other contains subunits with similarity to those found in the PAF1 complex involved in regulation of transcription in other eukaryotes. Thus, trypanosomatids utilize protein complexes similar to those used to control transcription termination in other eukaryotes and JBP3 appears to function as a hub linking these modules to base J, thereby enabling the parasites’ unique reliance on polycistronic transcription and post-transcriptional regulation of gene expression.

Post-transcriptional regulation of gene expression by Unr

2015 ◽  
Vol 43 (3) ◽  
pp. 323-327 ◽  
Author(s):  
Swagat Ray ◽  
Pól Ó Catnaigh ◽  
Emma C. Anderson
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Protein Interactions ◽  
Rna Binding ◽  
Regulation Of Gene Expression ◽  
Negative Regulator ◽  
Cellular Translation ◽  
Cellular Mrnas ◽  
Internal Initiation ◽  
Post Transcriptional Regulation

Unr (upstream of N-ras) is a eukaryotic RNA-binding protein that has a number of roles in the post-transcriptional regulation of gene expression. Originally identified as an activator of internal initiation of picornavirus translation, it has since been shown to act as an activator and inhibitor of cellular translation and as a positive and negative regulator of mRNA stability, regulating cellular processes such as mitosis and apoptosis. The different post-transcriptional functions of Unr depend on the identity of its mRNA and protein partners and can vary with cell type and changing cellular conditions. Recent high-throughput analyses of RNA–protein interactions indicate that Unr binds to a large subset of cellular mRNAs, suggesting that Unr may play a wider role in translational responses to cellular signals than previously thought.

scholarly journals System-wide analyses of the fission yeast poly(A)+ RNA interactome reveal insights into organisation and function of RNA-protein complexes

2019 ◽  
Author(s):  
Cornelia Kilchert ◽  
Tea Kecman ◽  
Emily Priest ◽  
Svenja Hester ◽  
Krzysztof Kus ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Protein Interactions ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Protein Complexes ◽  
Rna Degradation ◽  
Binding Activity ◽  
Rna Exosome ◽  
And Function ◽  
Rna Protein Complexes

AbstractProduction, function, and turnover of mRNA are orchestrated by multi-subunit machineries that play a central role in gene expression. Within these molecular machines, interactions with the target mRNA are mediated by RNA-binding proteins (RBPs), and the accuracy and dynamics of these RNA-protein interactions are essential for their function. Here, we show that fission yeast whole cell poly(A)+ RNA-protein crosslinking data provides system-wide information on the organisation and function of the RNA-protein complexes. We evaluate relative enrichment of cellular RBPs on poly(A)+ RNA to identify interactors with high RNA-binding activity and provide key information about the RNA-binding properties of large multi-protein complexes, such as the mRNA 3’ end processing machinery (cleavage and polyadenylation factor, CPF) and the RNA exosome. We demonstrate that different functional modules within CPF differ in their ability to interact with RNA. Importantly, we reveal that CPF forms additional contacts with RNA via the Fip1 subunit of the polyadenylation module and two subunits of the nuclease module. In addition, our data highlights the central role of the RNA helicase Mtl1 in RNA degradation by the exosome as mutations in Mtl1 lead to disengagement of the exosome from RNA. We examine how routes of substrate access to the complex are affected upon mutation of exosome subunits. Our results provide important insights into how different components of the exosome contribute to engagement of the complex with substrate RNA. Overall, our data uncover how multi-subunit cellular machineries interact with RNA, on a proteome-wide scale.

Predictive biomarkers in the treatment of acromegaly: a review of the literature

2020 ◽  
Vol 74 (6) ◽  
pp. 430-440
Author(s):  
E. G. Przhiyalkovskaya ◽  
Patimat O. Osmanova ◽  
Elizaveta O. Mamedova ◽  
Alexander S. Lutsenko ◽  
Zhanna E. Belaya ◽  
...  
Keyword(s):  
Somatostatin Receptors ◽  
Regulation Of Gene Expression ◽  
Predictive Biomarkers ◽  
Somatostatin Analogues ◽  
Epigenetic Biomarkers ◽  
Neurosurgical Treatment ◽  
Personalized Approach ◽  
The Individual ◽  
Post Transcriptional Regulation

In the era of personalized medicine treatment of acromegaly requires the individual selection of optimal treatment based on the measured parameters. Following the standard algorithm for the management of patients with acromegaly with the choice of neurosurgical treatment as the main and somatostatin analogues as the first line of drug therapy with ineffective surgery prevents the achievement of remission in patients resistant to these types of therapy. The introduction of predictive biomarkers in clinical practice will allow to achieve remission of the disease faster and reduce the financial costs of ineffective treatments. We collected information of possible predictive biomarkers in acromegaly from literature. This review presents data from studies of potential predictive biomarkers in different treatments of acromegaly. According to the analysis of publications, the greatest number of results is devoted to the prediction resistance to somatostatin analogues. Reliable biomarkers predicting the inefficiency of somatostatin analogues include low immunoexpression of somatostatin receptors type 2 and AIP protein, rarely granular type of pituitary adenoma and hyperintensive signal on T2-weighted images in magnetic resonance imaging of the pituitary gland. At the same time, the search for predictors of the effectiveness of pegvisomant is focused on the study of the receptor of growth hormone and opens up new opportunities for pharmacogenomic research. Thus, it is necessary to expand the search of predictive biomarkers for different methods of acromegalys treatment. It is especially important to identify biomarkers that do not require mandatory removal of the tumor. Of great interest is the study of epigenetic biomarkers, in particular miRNAs, which carry out post-transcriptional regulation of gene expression. The study of circulating blood microRNAs in acromegaly opens up prospects for the introduction of a personalized approach in the treatment of this disease.

scholarly journals Fructose modulates GLUT5 mRNA stability in differentiated Caco-2 cells: role of cAMP-signalling pathway and PABP (polyadenylated-binding protein)-interacting protein (Paip) 2

2003 ◽  
Vol 375 (1) ◽  
pp. 167-174 ◽  
Author(s):  
Florence GOUYON ◽  
Cercina ONESTO ◽  
Veronique DALET ◽  
Gilles PAGES ◽  
Armelle LETURQUE ◽  
...  
Keyword(s):  
Mrna Stability ◽  
Protein Complexes ◽  
Binding Protein ◽  
Interacting Protein ◽  
Camp Signalling ◽  
Camp Pathway ◽  
Post Transcriptional Regulation ◽  
Rna Protein Complexes ◽  
Camp Levels

In intestinal cells, levels of the fructose transporter GLUT5 are increased by glucose and to a greater extent by fructose. We investigated the mechanism by which fructose increases GLUT5 expression. In Caco-2 cells, fructose and glucose increased activity of the −2500/+41 GLUT5 promoter to the same extent. cAMP also activated the GLUT5 promoter. However, if a protein kinase A inhibitor was used to block cAMP signalling, extensive GLUT5 mRNA degradation was observed, with no change in basal transcription levels demonstrating the involvement of cAMP in GLUT5 mRNA stability. Indeed, the half-life of GLUT5 mRNA was correlated (R2=0.9913) with cellular cAMP levels. Fructose increased cAMP concentration more than glucose, accounting for the stronger effect of fructose when compared with that of glucose on GLUT5 production. We identified several complexes between GLUT5 3′-UTR RNA (where UTR stands for untranslated region) and cytosolic proteins that might participate in mRNA processing. Strong binding of a 140 kDa complex I was observed in sugar-deprived cells, with levels of binding lower in the presence of fructose and glucose by factors of 12 and 6 respectively. This may account for differences in the effects of fructose and glucose. In contrast, the amounts of two complexes of 96 and 48 kDa increased equally after stimulation with either glucose or fructose. Finally, PABP (polyadenylated-binding protein)-interacting protein 2, a destabilizing partner of PABP, was identified as a component of GLUT5 3′-UTR RNA–protein complexes. We conclude that the post-transcriptional regulation of GLUT5 by fructose involves increases in mRNA stability mediated by the cAMP pathway and Paip2 (PABP-interacting protein 2) binding.

scholarly journals Lineberger Comprehensive Cancer Center University of North Carolina

1993 ◽  
Vol 1 (5) ◽  
pp. 9-13
Keyword(s):  
Electron Microscopy ◽  
North Carolina ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Nobel Prize Winner ◽  
Comprehensive Cancer Center ◽  
Cancer Center ◽  
University Of North Carolina ◽  
Dna And Rna ◽  
Rna Protein Complexes

The electron microscope facility at the Lineberger Comprehensive Cancer Center (LCCC), at the University of North Carolina, is a world leader in the analysis and visualization of DNA- and RNA-protein complexes. Established by Dr. Jack Griffith in 1978, the facility houses the only laboratory in the U.S. specifically dedicated to visualizing DNA- and RNA-protein interactions and is frequently called upon by research institutions and organizations to provide breakthrough scientific findings. For instance, Dr. Griffith, in collaboration with Dr. Paul Englund at John Hopkins University, was the first to visualize DNA molecules with unusual curvatures; and, working with Dr. Mark Ptashne and Dr. Tom Maniatis at Harvard, was the first to visualize the looping of DNA and splicesomes. Presently, the lab is working with Nobel Prize winner Dr. Arthur Kornberg at Stanford University to understand how DNA replicates.According to Dr. Griffith, LCCC's electron microscopy system is part of a unique scientific approach to studying DNA- and RNA-protein complexes. The approach integrates electron microscopy with conventional molecular biologic and biochemical research methods.

scholarly journals Characterization of major chromatin assembly proteins in tetrahymena thermophile using proeomic and molecular evolutionary analyses

2021 ◽  
Author(s):  
Syed N Shah
Keyword(s):  
Protein Interactions ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Regulation Of Gene Expression ◽  
Chromatin Assembly ◽  
Histone Chaperones ◽  
Protein Protein Interactions ◽  
Selective Forces ◽  
Assembly Proteins

Histones H3/H4 are deposited onto DNA in a replication-dependent or independent fashion by the CAF1 and HIRA protein complexes. Despite the identification of these protein complexes, mechanistic details remain unclear. Recently, we showed that in T. thermophila histone chaperones Nrp1, Asf1 and the Impβ6 importin function together to transport newly synthesized H3/H4 from the cytoplasm to the nucleus. To characterize chromatin assembly proteins in T.thermophila, I used affinity purification combined with mass spectrometry to identify protein-protein interactions of Nrp1, Cac2 subunit of CAF1, HIRA and histone modifying Hat1-complex in T. thermophila. I found that the three-subunit T.thermophila CAF1 complex interacts with Casein Kinase 2 (CKII), possibly accounting for previously reported human CAF1phosphorylation. I also found that Hat2 subunit of HAT1 complex is also shared by CAF1 complex as its Cac3 subunit. This suggests that Hat2/Cac3 might exist in two separate pools of protein complexes. Remarkably, proteomic analysis of Hat2/Cac3 in turn revealed that it forms several complexes with other proteins including SIN3, RXT3, LIN9 and TESMIN, all of which have known roles in the regulation of gene expression. Finally, I asked how selective forces might have impacted on the function of proteins involved in H3/H4 transport. Focusing on NASP which possesses several TPR motifs, I showed that its protein-protein interactions are conserved in T. thermophila. Using molecular evolutionary methods I show that different TPRs in NASP evolve at different rates possibly accounting for the functional diversity observed among different family members.

scholarly journals Sampling native-like structures of RNA-protein complexes through Rosetta folding and docking

2018 ◽  
Author(s):  
Kalli Kappel ◽  
Rhiju Das
Keyword(s):  
Conformational Changes ◽  
Posttranscriptional Regulation ◽  
De Novo ◽  
Protein Complexes ◽  
Regulation Of Gene Expression ◽  
Low Resolution ◽  
Human Telomerase ◽  
Nucleotide Resolution ◽  
Rna Protein Complexes ◽  
Resolution Data

AbstractRNA-protein complexes underlie numerous cellular processes including translation, splicing, and posttranscriptional regulation of gene expression. The structures of these complexes are crucial to their functions but often elude high-resolution structure determination. Computational methods are needed that can integrate low-resolution data for RNA-protein complexes while modeling de novo the large conformational changes of RNA components upon complex formation. To address this challenge, we describe a Rosetta method called RNP-denovo to simultaneously fold and dock RNA to a protein surface. On a benchmark set of structurally diverse RNA-protein complexes that are not solvable with prior strategies, this fold-and-dock method consistently sampled native-like structures with better than nucleotide resolution. We revisited three past blind modeling challenges in which previous methods gave poor results: human telomerase, an RNA methyltransferase with a ribosomal RNA domain, and the spliceosome. When coupled with the same sparse FRET, cross-linking, and functional data used in previous work, RNP-denovo gave models with significantly improved accuracy. These results open a route to computationally modeling global folds of RNA-protein complexes from low-resolution data.

scholarly journals mSphere of Influence: Modifying an Old Method To Study RNA-Protein Interactions

mSphere ◽  
2019 ◽  
Vol 4 (5) ◽  
Author(s):  
Calvin Tiengwe
Keyword(s):  
Gene Regulation ◽  
Trypanosoma Brucei ◽  
Protein Interactions ◽  
Iron Homeostasis ◽  
Protein Complexes ◽  
Global Scale ◽  
Content Type ◽  
Posttranscriptional Gene Regulation ◽  
Guanidinium Thiocyanate ◽  
Rna Protein Complexes

ABSTRACT Calvin Tiengwe works on posttranscriptional gene regulation and iron homeostasis in the parasitic protozoan Trypanosoma brucei. In this mSphere of Influence article, he reflects on how the paper “Comprehensive identification of RNA-protein interactions in any organism using orthogonal organic phase separation (OOPS)” by Queiroz et al. (Nat Biotechnol 37:169–178, 2019, https://doi.org/10.1038/s41587-018-0001-2) influenced his research by providing a tool to capture RNA-protein complexes on a global scale using acid guanidinium thiocyanate-phenol-chloroform (AGPC), an old method hitherto applied for RNA, DNA, or protein purification.

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