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19 pages, 3510 KiB

Open AccessArticle

Stress Granule Assembly Can Facilitate but Is Not Required for TDP-43 Cytoplasmic Aggregation

by Nikita Fernandes, Luke Nero, Shawn M. Lyons, Pavel Ivanov, Telsa M. Mittelmeier, Timothy A. Bolger and J. Ross Buchan

Biomolecules 2020, 10(10), 1367; https://doi.org/10.3390/biom10101367 - 25 Sep 2020

Cited by 23 | Viewed by 5380 | Correction

Abstract

Stress granules (SGs) are hypothesized to facilitate TAR DNA-binding protein 43 (TDP-43) cytoplasmic mislocalization and aggregation, which may underly amyotrophic lateral sclerosis pathology. However, much data for this hypothesis is indirect. Additionally, whether P-bodies (PBs; related mRNA-protein granules) affect TDP-43 phenotypes is unclear. [...] Read more.

Stress granules (SGs) are hypothesized to facilitate TAR DNA-binding protein 43 (TDP-43) cytoplasmic mislocalization and aggregation, which may underly amyotrophic lateral sclerosis pathology. However, much data for this hypothesis is indirect. Additionally, whether P-bodies (PBs; related mRNA-protein granules) affect TDP-43 phenotypes is unclear. Here, we determine that induction of TDP-43 expression in yeast results in the accumulation of SG-like foci that in >90% of cases become the sites where TDP-43 cytoplasmic foci first appear. Later, TDP-43 foci associate less with SGs and more with PBs, though independent TDP-43 foci also accumulate. However, depleting or over-expressing yeast SG and PB proteins reveals no consistent trend between SG or PB assembly and TDP-43 foci formation, toxicity or protein abundance. In human cells, immunostaining endogenous TDP-43 with different TDP-43 antibodies reveals distinct localization and aggregation behaviors. Following acute arsenite stress, all phospho-TDP-43 foci colocalize with SGs. Finally, formation of TDP-43 cytoplasmic foci following low-dose chronic arsenite stress is impaired, but not completely blocked, in G3BP1/2ΔΔ cells. Collectively, our data suggest that SG and PB assembly may facilitate TDP-43 cytoplasmic localization and aggregation but are likely not essential for these events. Full article

(This article belongs to the Special Issue Ribonucleoprotein Particles (RNPs): From Structure to Function)

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19 pages, 1708 KiB

Open AccessArticle

CTELS: A Cell-Free System for the Analysis of Translation Termination Rate

by Kseniya A. Lashkevich, Valeriya I. Shlyk, Artem S. Kushchenko, Vadim N. Gladyshev, Elena Z. Alkalaeva and Sergey E. Dmitriev

Biomolecules 2020, 10(6), 911; https://doi.org/10.3390/biom10060911 - 16 Jun 2020

Cited by 12 | Viewed by 4304

Abstract

Translation termination is the final step in protein biosynthesis when the synthesized polypeptide is released from the ribosome. Understanding this complex process is important for treatment of many human disorders caused by nonsense mutations in important genes. Here, we present a new method [...] Read more.

Translation termination is the final step in protein biosynthesis when the synthesized polypeptide is released from the ribosome. Understanding this complex process is important for treatment of many human disorders caused by nonsense mutations in important genes. Here, we present a new method for the analysis of translation termination rate in cell-free systems, CTELS (for C-terminally extended luciferase-based system). This approach was based on a continuously measured luciferase activity during in vitro translation reaction of two reporter mRNA, one of which encodes a C-terminally extended luciferase. This extension occupies a ribosomal polypeptide tunnel and lets the completely synthesized enzyme be active before translation termination occurs, i.e., when it is still on the ribosome. In contrast, luciferase molecule without the extension emits light only after its release. Comparing the translation dynamics of these two reporters allows visualization of a delay corresponding to the translation termination event. We demonstrated applicability of this approach for investigating the effects of cis- and trans-acting components, including small molecule inhibitors and read-through inducing sequences, on the translation termination rate. With CTELS, we systematically assessed negative effects of decreased 3′ UTR length, specifically on termination. We also showed that blasticidin S implements its inhibitory effect on eukaryotic translation system, mostly by affecting elongation, and that an excess of eRF1 termination factor (both the wild-type and a non-catalytic AGQ mutant) can interfere with elongation. Analysis of read-through mechanics with CTELS revealed a transient stalling event at a “leaky” stop codon context, which likely defines the basis of nonsense suppression. Full article

(This article belongs to the Special Issue Ribonucleoprotein Particles (RNPs): From Structure to Function)

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12 pages, 1727 KiB

Open AccessArticle

Crystal Structure of a Variant PAM2 Motif of LARP4B Bound to the MLLE Domain of PABPC1

by Clemens Grimm, Jann-Patrick Pelz, Cornelius Schneider, Katrin Schäffler and Utz Fischer

Biomolecules 2020, 10(6), 872; https://doi.org/10.3390/biom10060872 - 06 Jun 2020

Cited by 6 | Viewed by 3279

Abstract

Eukaryotic cells determine the protein output of their genetic program by regulating mRNA transcription, localization, translation and turnover rates. This regulation is accomplished by an ensemble of RNA-binding proteins (RBPs) that bind to any given mRNA, thus forming mRNPs. Poly(A) binding proteins (PABPs) [...] Read more.

Eukaryotic cells determine the protein output of their genetic program by regulating mRNA transcription, localization, translation and turnover rates. This regulation is accomplished by an ensemble of RNA-binding proteins (RBPs) that bind to any given mRNA, thus forming mRNPs. Poly(A) binding proteins (PABPs) are prominent members of virtually all mRNPs that possess poly(A) tails. They serve as multifunctional scaffolds, allowing the recruitment of diverse factors containing a poly(A)-interacting motif (PAM) into mRNPs. We present the crystal structure of the variant PAM motif (termed PAM2w) in the N-terminal part of the positive translation factor LARP4B, which binds to the MLLE domain of the poly(A) binding protein C1 cytoplasmic 1 (PABPC1). The structural analysis, along with mutational studies in vitro and in vivo, uncovered a new mode of interaction between PAM2 motifs and MLLE domains. Full article

(This article belongs to the Special Issue Ribonucleoprotein Particles (RNPs): From Structure to Function)

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21 pages, 3351 KiB

Open AccessEditor’s ChoiceArticle

Discovering the RNA-Binding Proteome of Plant Leaves with an Improved RNA Interactome Capture Method

by Marcel Bach-Pages, Felix Homma, Jiorgos Kourelis, Farnusch Kaschani, Shabaz Mohammed, Markus Kaiser, Renier A. L. van der Hoorn, Alfredo Castello and Gail M. Preston

Biomolecules 2020, 10(4), 661; https://doi.org/10.3390/biom10040661 - 24 Apr 2020

Cited by 55 | Viewed by 14123

Abstract

RNA-binding proteins (RBPs) play a crucial role in regulating RNA function and fate. However, the full complement of RBPs has only recently begun to be uncovered through proteome-wide approaches such as RNA interactome capture (RIC). RIC has been applied to various cell lines [...] Read more.

RNA-binding proteins (RBPs) play a crucial role in regulating RNA function and fate. However, the full complement of RBPs has only recently begun to be uncovered through proteome-wide approaches such as RNA interactome capture (RIC). RIC has been applied to various cell lines and organisms, including plants, greatly expanding the repertoire of RBPs. However, several technical challenges have limited the efficacy of RIC when applied to plant tissues. Here, we report an improved version of RIC that overcomes the difficulties imposed by leaf tissue. Using this improved RIC method in Arabidopsis leaves, we identified 717 RBPs, generating a deep RNA-binding proteome for leaf tissues. While 75% of these RBPs can be linked to RNA biology, the remaining 25% were previously not known to interact with RNA. Interestingly, we observed that a large number of proteins related to photosynthesis associate with RNA in vivo, including proteins from the four major photosynthetic supercomplexes. As has previously been reported for mammals, a large proportion of leaf RBPs lack known RNA-binding domains, suggesting unconventional modes of RNA binding. We anticipate that this improved RIC method will provide critical insights into RNA metabolism in plants, including how cellular RBPs respond to environmental, physiological and pathological cues. Full article

(This article belongs to the Special Issue Ribonucleoprotein Particles (RNPs): From Structure to Function)

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20 pages, 4836 KiB

Open AccessArticle

Human vtRNA1-1 Levels Modulate Signaling Pathways and Regulate Apoptosis in Human Cancer Cells

by Lisamaria Bracher, Iolanda Ferro, Carlos Pulido-Quetglas, Marc-David Ruepp, Rory Johnson and Norbert Polacek

Biomolecules 2020, 10(4), 614; https://doi.org/10.3390/biom10040614 - 16 Apr 2020

Cited by 20 | Viewed by 3986

Abstract

Regulatory non-protein coding RNAs perform a remarkable variety of complex biological functions. Previously, we demonstrated a role of the human non-coding vault RNA1-1 (vtRNA1-1) in inhibiting intrinsic and extrinsic apoptosis in several cancer cell lines. Yet on the molecular level, the function of [...] Read more.

Regulatory non-protein coding RNAs perform a remarkable variety of complex biological functions. Previously, we demonstrated a role of the human non-coding vault RNA1-1 (vtRNA1-1) in inhibiting intrinsic and extrinsic apoptosis in several cancer cell lines. Yet on the molecular level, the function of the vtRNA1-1 is still not fully clear. Here, we created HeLa knock-out cell lines revealing that prolonged starvation triggers elevated levels of apoptosis in the absence of vtRNA1-1 but not in vtRNA1-3 knock-out cells. Next-generation deep sequencing of the mRNome identified the PI3K/Akt pathway and the ERK1/2 MAPK cascade, two prominent signaling axes, to be misregulated in the absence of vtRNA1-1 during starvation-mediated cell death conditions. Expression of vtRNA1-1 mutants identified a short stretch of 24 nucleotides of the vtRNA1-1 central domain as being essential for successful maintenance of apoptosis resistance. This study describes a cell signaling-dependent contribution of the human vtRNA1-1 to starvation-induced programmed cell death. Full article

(This article belongs to the Special Issue Ribonucleoprotein Particles (RNPs): From Structure to Function)

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Review

Jump to: Research

32 pages, 2618 KiB

Open AccessReview

Dynamic mRNP Remodeling in Response to Internal and External Stimuli

by Kathi Zarnack, Sureshkumar Balasubramanian, Michael P. Gantier, Vladislav Kunetsky, Michael Kracht, M. Lienhard Schmitz and Katja Sträßer

Biomolecules 2020, 10(9), 1310; https://doi.org/10.3390/biom10091310 - 11 Sep 2020

Cited by 12 | Viewed by 5175

Abstract

Signal transduction and the regulation of gene expression are fundamental processes in every cell. RNA-binding proteins (RBPs) play a key role in the post-transcriptional modulation of gene expression in response to both internal and external stimuli. However, how signaling pathways regulate the assembly [...] Read more.

Signal transduction and the regulation of gene expression are fundamental processes in every cell. RNA-binding proteins (RBPs) play a key role in the post-transcriptional modulation of gene expression in response to both internal and external stimuli. However, how signaling pathways regulate the assembly of RBPs with mRNAs remains largely unknown. Here, we summarize observations showing that the formation and composition of messenger ribonucleoprotein particles (mRNPs) is dynamically remodeled in space and time by specific signaling cascades and the resulting post-translational modifications. The integration of signaling events with gene expression is key to the rapid adaptation of cells to environmental changes and stress. Only a combined approach analyzing the signal transduction pathways and the changes in post-transcriptional gene expression they cause will unravel the mechanisms coordinating these important cellular processes. Full article

(This article belongs to the Special Issue Ribonucleoprotein Particles (RNPs): From Structure to Function)

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27 pages, 2410 KiB

Open AccessReview

Single and Combined Methods to Specifically or Bulk-Purify RNA–Protein Complexes

by Roosje Van Ende, Sam Balzarini and Koen Geuten

Biomolecules 2020, 10(8), 1160; https://doi.org/10.3390/biom10081160 - 07 Aug 2020

Cited by 13 | Viewed by 4797

Abstract

The ribonome interconnects the proteome and the transcriptome. Specific biology is situated at this interface, which can be studied in bulk using omics approaches or specifically by targeting an individual protein or RNA species. In this review, we focus on both RNA- and [...] Read more.

The ribonome interconnects the proteome and the transcriptome. Specific biology is situated at this interface, which can be studied in bulk using omics approaches or specifically by targeting an individual protein or RNA species. In this review, we focus on both RNA- and ribonucleoprotein-(RNP) centric methods. These methods can be used to study the dynamics of the ribonome in response to a stimulus or to identify the proteins that interact with a specific RNA species. The purpose of this review is to provide and discuss an overview of strategies to cross-link RNA to proteins and the currently available RNA- and RNP-centric approaches to study RNPs. We elaborate on some major challenges common to most methods, involving RNP yield, purity and experimental cost. We identify the origin of these difficulties and propose to combine existing approaches to overcome these challenges. The solutions provided build on the recently developed organic phase separation protocols, such as Cross-Linked RNA eXtraction (XRNAX), orthogonal organic phase separation (OOPS) and Phenol-Toluol extraction (PTex). Full article

(This article belongs to the Special Issue Ribonucleoprotein Particles (RNPs): From Structure to Function)

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34 pages, 1376 KiB

Open AccessEditor’s ChoiceReview

UPF1-Mediated RNA Decay—Danse Macabre in a Cloud

by Daria Lavysh and Gabriele Neu-Yilik

Biomolecules 2020, 10(7), 999; https://doi.org/10.3390/biom10070999 - 04 Jul 2020

Cited by 19 | Viewed by 5798

Abstract

Nonsense-mediated RNA decay (NMD) is the prototype example of a whole family of RNA decay pathways that unfold around a common central effector protein called UPF1. While NMD in yeast appears to be a linear pathway, NMD in higher eukaryotes is a multifaceted [...] Read more.

Nonsense-mediated RNA decay (NMD) is the prototype example of a whole family of RNA decay pathways that unfold around a common central effector protein called UPF1. While NMD in yeast appears to be a linear pathway, NMD in higher eukaryotes is a multifaceted phenomenon with high variability with respect to substrate RNAs, degradation efficiency, effector proteins and decay-triggering RNA features. Despite increasing knowledge of the mechanistic details, it seems ever more difficult to define NMD and to clearly distinguish it from a growing list of other UPF1-mediated RNA decay pathways (UMDs). With a focus on mammalian NMD, we here critically examine the prevailing NMD models and the gaps and inconsistencies in these models. By exploring the minimal requirements for NMD and other UMDs, we try to elucidate whether they are separate and definable pathways, or rather variations of the same phenomenon. Finally, we suggest that the operating principle of the UPF1-mediated decay family could be considered similar to that of a computing cloud providing a flexible infrastructure with rapid elasticity and dynamic access according to specific user needs. Full article

(This article belongs to the Special Issue Ribonucleoprotein Particles (RNPs): From Structure to Function)

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18 pages, 1408 KiB

Open AccessReview

TOP mRNPs: Molecular Mechanisms and Principles of Regulation

by Eric Cockman, Paul Anderson and Pavel Ivanov

Biomolecules 2020, 10(7), 969; https://doi.org/10.3390/biom10070969 - 27 Jun 2020

Cited by 36 | Viewed by 6280

Abstract

The cellular response to changes in the surrounding environment and to stress requires the coregulation of gene networks aiming to conserve energy and resources. This is often achieved by downregulating protein synthesis. The 5’ Terminal OligoPyrimidine (5’ TOP) motif-containing mRNAs, which encode proteins [...] Read more.

The cellular response to changes in the surrounding environment and to stress requires the coregulation of gene networks aiming to conserve energy and resources. This is often achieved by downregulating protein synthesis. The 5’ Terminal OligoPyrimidine (5’ TOP) motif-containing mRNAs, which encode proteins that are essential for protein synthesis, are the primary targets of translational control under stress. The TOP motif is a cis-regulatory RNA element that begins directly after the m7G cap structure and contains the hallmark invariant 5’-cytidine followed by an uninterrupted tract of 4–15 pyrimidines. Regulation of translation via the TOP motif coordinates global protein synthesis with simultaneous co-expression of the protein components required for ribosome biogenesis. In this review, we discuss architecture of TOP mRNA-containing ribonucleoprotein complexes, the principles of their assembly, and the modes of regulation of TOP mRNA translation. Full article

(This article belongs to the Special Issue Ribonucleoprotein Particles (RNPs): From Structure to Function)

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43 pages, 3347 KiB

Open AccessReview

Emerging Roles of RNA 3′-end Cleavage and Polyadenylation in Pathogenesis, Diagnosis and Therapy of Human Disorders

by Jamie Nourse, Stefano Spada and Sven Danckwardt

Biomolecules 2020, 10(6), 915; https://doi.org/10.3390/biom10060915 - 17 Jun 2020

Cited by 41 | Viewed by 8441

Abstract

A crucial feature of gene expression involves RNA processing to produce 3′ ends through a process termed 3′ end cleavage and polyadenylation (CPA). This ensures the nascent RNA molecule can exit the nucleus and be translated to ultimately give rise to a protein [...] Read more.

A crucial feature of gene expression involves RNA processing to produce 3′ ends through a process termed 3′ end cleavage and polyadenylation (CPA). This ensures the nascent RNA molecule can exit the nucleus and be translated to ultimately give rise to a protein which can execute a function. Further, alternative polyadenylation (APA) can produce distinct transcript isoforms, profoundly expanding the complexity of the transcriptome. CPA is carried out by multi-component protein complexes interacting with multiple RNA motifs and is tightly coupled to transcription, other steps of RNA processing, and even epigenetic modifications. CPA and APA contribute to the maintenance of a multitude of diverse physiological processes. It is therefore not surprising that disruptions of CPA and APA can lead to devastating disorders. Here, we review potential CPA and APA mechanisms involving both loss and gain of function that can have tremendous impacts on health and disease. Ultimately we highlight the emerging diagnostic and therapeutic potential CPA and APA offer. Full article

(This article belongs to the Special Issue Ribonucleoprotein Particles (RNPs): From Structure to Function)

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15 pages, 1692 KiB

Open AccessReview

Human Telomerase RNA: Telomerase Component or More?

by Maria Rubtsova and Olga Dontsova

Biomolecules 2020, 10(6), 873; https://doi.org/10.3390/biom10060873 - 06 Jun 2020

Cited by 15 | Viewed by 4926

Abstract

Telomerase is a ribonucleoprotein complex that maintains the lengths of telomeres. Most studies of telomerase function have focused on the involvement of telomerase activation in the immortalization of cancer cells and cellular rejuvenation. However, some studies demonstrated that the results do not meet [...] Read more.

Telomerase is a ribonucleoprotein complex that maintains the lengths of telomeres. Most studies of telomerase function have focused on the involvement of telomerase activation in the immortalization of cancer cells and cellular rejuvenation. However, some studies demonstrated that the results do not meet expectations for telomerase action in telomere maintenance. Recent results give reason to think that major telomerase components—the reverse transcriptase protein subunit and telomerase RNA—may participate in many cellular processes, including the regulation of apoptosis and autophagy, cell survival, pro-proliferative effects, regulation of gene expression, and protection against oxidative stress. However, the difficulties faced by scientist when researching telomerase component functions often reduce confidence in the minor effects observed in experiments. In this review, we focus on the analysis of the functions of telomerase components (paying more attention to the telomerase RNA component), both as a complex and as independent components, providing effects that are not associated with telomerase activity and telomere length maintenance. Despite the fact that the data on alternative roles of telomerase components look illusory, it would be wrong to completely reject the possibility of their involvement in other biological processes excluded from research/discussion. Investigations to improve the understanding of every aspect of the functioning of telomerase components will provide the basis for a more precise development of approaches to regulate cellular homeostasis, which is important for carcinogenesis and aging. Full article

(This article belongs to the Special Issue Ribonucleoprotein Particles (RNPs): From Structure to Function)

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17 pages, 2724 KiB

Open AccessReview

A Day in the Life of the Exon Junction Complex

by Lena P. Schlautmann and Niels H. Gehring

Biomolecules 2020, 10(6), 866; https://doi.org/10.3390/biom10060866 - 05 Jun 2020

Cited by 48 | Viewed by 8451

Abstract

The exon junction complex (EJC) is an abundant messenger ribonucleoprotein (mRNP) component that is assembled during splicing and binds to mRNAs upstream of exon-exon junctions. EJCs accompany the mRNA during its entire life in the nucleus and the cytoplasm and communicate the information [...] Read more.

The exon junction complex (EJC) is an abundant messenger ribonucleoprotein (mRNP) component that is assembled during splicing and binds to mRNAs upstream of exon-exon junctions. EJCs accompany the mRNA during its entire life in the nucleus and the cytoplasm and communicate the information about the splicing process and the position of introns. Specifically, the EJC’s core components and its associated proteins regulate different steps of gene expression, including pre-mRNA splicing, mRNA export, translation, and nonsense-mediated mRNA decay (NMD). This review summarizes the most important functions and main protagonists in the life of the EJC. It also provides an overview of the latest findings on the assembly, composition and molecular activities of the EJC and presents them in the chronological order, in which they play a role in the EJC’s life cycle. Full article

(This article belongs to the Special Issue Ribonucleoprotein Particles (RNPs): From Structure to Function)

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28 pages, 2707 KiB

Open AccessReview

snoRNPs: Functions in Ribosome Biogenesis

by Sandeep Ojha, Sulochan Malla and Shawn M. Lyons

Biomolecules 2020, 10(5), 783; https://doi.org/10.3390/biom10050783 - 18 May 2020

Cited by 58 | Viewed by 8703

Abstract

Ribosomes are perhaps the most critical macromolecular machine as they are tasked with carrying out protein synthesis in cells. They are incredibly complex structures composed of protein components and heavily chemically modified RNAs. The task of assembling mature ribosomes from their component parts [...] Read more.

Ribosomes are perhaps the most critical macromolecular machine as they are tasked with carrying out protein synthesis in cells. They are incredibly complex structures composed of protein components and heavily chemically modified RNAs. The task of assembling mature ribosomes from their component parts consumes a massive amount of energy and requires greater than 200 assembly factors. Among the most critical of these are small nucleolar ribonucleoproteins (snoRNPs). These are small RNAs complexed with diverse sets of proteins. As suggested by their name, they localize to the nucleolus, the site of ribosome biogenesis. There, they facilitate multiple roles in ribosomes biogenesis, such as pseudouridylation and 2′-O-methylation of ribosomal (r)RNA, guiding pre-rRNA processing, and acting as molecular chaperones. Here, we reviewed their activity in promoting the assembly of ribosomes in eukaryotes with regards to chemical modification and pre-rRNA processing. Full article

(This article belongs to the Special Issue Ribonucleoprotein Particles (RNPs): From Structure to Function)

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30 pages, 978 KiB

Open AccessReview

Y-Box Binding Proteins in mRNP Assembly, Translation, and Stability Control

by Daria Mordovkina, Dmitry N. Lyabin, Egor A. Smolin, Ekaterina M. Sogorina, Lev P. Ovchinnikov and Irina Eliseeva

Biomolecules 2020, 10(4), 591; https://doi.org/10.3390/biom10040591 - 11 Apr 2020

Cited by 70 | Viewed by 5730

Abstract

Y-box binding proteins (YB proteins) are DNA/RNA-binding proteins belonging to a large family of proteins with the cold shock domain. Functionally, these proteins are known to be the most diverse, although the literature hardly offers any molecular mechanisms governing their activities in the [...] Read more.

Y-box binding proteins (YB proteins) are DNA/RNA-binding proteins belonging to a large family of proteins with the cold shock domain. Functionally, these proteins are known to be the most diverse, although the literature hardly offers any molecular mechanisms governing their activities in the cell, tissue, or the whole organism. This review describes the involvement of YB proteins in RNA-dependent processes, such as mRNA packaging into mRNPs, mRNA translation, and mRNA stabilization. In addition, recent data on the structural peculiarities of YB proteins underlying their interactions with nucleic acids are discussed. Full article

(This article belongs to the Special Issue Ribonucleoprotein Particles (RNPs): From Structure to Function)

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