Cold Shock Domain Family Members YB-1 and MSY4 Share Essential Functions during Murine Embryogenesis

Lu, Zhi Hong; Books, Jason T.; Ley, Timothy J.

doi:10.1128/mcb.01196-06

Cited by 74 publications

(81 citation statements)

References 27 publications

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“…In addition to the known ZBP1 (also known as IMP-1 or IGF2BP1) we found 5 other proteins that bound with significant SILAC ratios: the binding of IMP-3 can readily be rationalized by its close homology to IMP-1 (24) and YB1 has previously been shown to be part of IMP-1 RNP granules (25). Interestingly, CSDA (also known as DBPA) is closely homologous to YB1 (26) and both proteins contain a cold shock domain. IMP-1/IMP-3 and YB1/CSDA are closely related proteins, strongly suggesting biological relevance.…”

Section: Detection Of Rbps For H4 Mrna Stem Loop and Zipcode Rna Sequmentioning

confidence: 84%

Unbiased RNA–protein interaction screen by quantitative proteomics

Butter

Scheibe

Mörl

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

130

115

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Mass spectrometry (MS)-based quantitative interaction proteomics has successfully elucidated specific protein-protein, DNA-protein, and small molecule-protein interactions. Here, we developed a gelfree, sensitive, and scalable technology that addresses the important area of RNA-protein interactions. Using aptamer-tagged RNA as bait, we captured RNA-interacting proteins from stable isotope labeling by amino acids in cell culture (SILAC)-labeled mammalian cell extracts and analyzed them by high-resolution, quantitative MS. Binders specific to the RNA sequence were distinguished from background by their isotope ratios between bait and control. We demonstrated the approach by retrieving known and novel interaction partners for the HuR interaction motif, H4 stem loop, ''zipcode'' sequence, tRNA, and a bioinformatically-predicted RNA fold in DGCR-8/Pasha mRNA. In all experiments we unambiguously identified known interaction partners by a single affinity purification step. The 5 region of the mRNA of DGCR-8/Pasha, a component of the microprocessor complex, specifically interacts with components of the translational machinery, suggesting that it contains an internal ribosome entry site.quantitative mass spectrometry ͉ ribonucleoprotein ͉ RNA-binding proteins ͉ HuR ͉ internal ribosome entry site R ibonucleic acid (RNA) is increasingly recognized for its diversity of cellular functions ranging from its classical roles as structural component in complexes such as the ribosome and its role as the central intermediate in gene expression to recentlydiscovered, critical roles in gene regulation (1). Analysis of RNA can efficiently be performed by hybridization or sequencing-based methods; however, in the cellular environment RNA is associated with RNA-binding proteins (RBPs) forming functional ribonucleoprotein (RNP) complexes. These proteins are essential to the function of RNPs but have been studied much less.Mass spectrometry (MS)-based proteomics has become a widespread tool for studying complex mixtures of proteins at high sensitivity (2, 3). High-resolution and high-accuracy technologies have recently been introduced at a large scale (4), and entire proteomes can now be quantified (5). Quantitative proteomics has also emerged as a powerful tool for detecting specific binding of proteins to baits by an unbiased proteome-wide screen using peptides and proteins (reviewed in ref. 6) and DNA (7, 8) as baits. These screens have provided candidates that subsequently proved to be of physiological importance in vivo (7, 9, 10). However, application of this technique to detect RBPs has not been reported to our knowledge.In one technology of quantitative proteomics, stable isotope labeling of amino acids in cell culture (SILAC), 2 cell populations are metabolically encoded with either 13 C 6 (heavy) amino acids or 12 C 6 (light) amino acids (11,12). Background proteins occur equally in control and bait eluate, and the SILAC peptide pairs consequently have a 1:1 intensity ratio. Specific binders to the bait have heavy/light ratio si...

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Section: Detection Of Rbps For H4 Mrna Stem Loop and Zipcode Rna Sequmentioning

confidence: 84%

Unbiased RNA–protein interaction screen by quantitative proteomics

Butter

Scheibe

Mörl

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

130

115

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“…However, YBX2 appears to be the critical isoform because the Ybx2-null mutation totally blocks spermatid development, whereas the Ybx3-null mutation has slight effects on spermatid development and male fertility (Lu et al 2006, Yang et al 2007. The more deleterious phenotype of the Ybx2-null mutation is likely related with observations that YBX2 is the predominant RBP in testis free-mRNPs (Herbert & Hecht 1999), and is much more abundant than YBX3L.…”

Section: Discussionmentioning

confidence: 98%

Mechanisms of translational repression of the Smcp mRNA in round spermatids

Cullinane¹,

Chowdhury²,

Kleene³

2015

Reproduction

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The protamine 1 (Prm1) and sperm mitochondria-associated, cysteine-rich protein (Smcp) mRNAs exemplify a widespread pattern of mRNA-specific regulation of mRNA translation in post-meiotic spermatogenic cells, spermatids. Both mRNAs are transcribed and initially stored in free-mRNPs in early spermatids, and translated on polysomes in late spermatids. In this study, we demonstrate that the 5 0 and 3 0 -UTRs and the 3 0 terminus of the Smcp 3 0 -UTR are required for normal repression of the Smcp mRNA in transgenic mice. RNA affinity chromatography and mass spectrometry sequencing identified Y-box protein 2 (YBX2/MSY2) as the major protein that interacts with the 3 0 terminus of the Smcp 3 0 -UTR and a Y-box recognition sequence, GCCACCU, in the translation control element that is necessary for Prm1 mRNA repression. Depletion of YBX2 in Ybx2-null mice prematurely activates Prm1 and Smcp mRNA translation in early spermatids. Fluorescent in situ hybridization reveals that the Smcp intron, the Smcp mRNA, and both Smcp-Gfp transgenic mRNAs are strongly concentrated in the chromatoid body, and that theYbx2-null mutation does not eliminate the Smcp mRNA from the chromatoid body. This and previous findings suggest that the Smcp pre-mRNA is spliced and associates with YBX2 in the chromatoid body, and that repressed free-mRNPs are stored in the general cytoplasm. As YBX2 is the predominant protein in testis free-mRNPs, it likely represses many mRNAs in early spermatids. The mechanisms by which YBX2 represses the Smcp and Prm1 mRNAs are relevant to reproductive medicine because mutations in the human YBX2 gene correlate with abnormal protamine expression and male infertility.

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“…Y-box proteins have been described in bacteria, plants, and animals, and all of them contain a "cold shock" domain (CSD). Three members of this family were identified in vertebrates (45,46), but only YBX1 is expressed ubiquitously in adults. In vertebrate cells, the Y-box proteins are comprised of three domains: an alanine-and proline-rich N-terminal domain, followed by a CSD domain and a C-terminal domain that consists of repeated sequences of predominantly basic or acidic amino acids (B/A repeat) (32).…”

Section: Vol 82 2008 Nsp3-specific Complexes In Virus Replication 1mentioning

confidence: 99%

Different Types of nsP3-Containing Protein Complexes in Sindbis Virus-Infected Cells

Gorchakov

Garmashova

Frolova

et al. 2008

J Virol

129

190

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Alphaviruses represent a serious public health threat and cause a wide variety of diseases, ranging from severe encephalitis, which can result in death or neurological sequelae, to mild infection, characterized by fever, skin rashes, and arthritis. In the infected cells, alphaviruses express only four nonstructural proteins, which function in the synthesis of virus-specific RNAs and in modification of the intracellular environment. The results of our study suggest that Sindbis virus (SINV) infection in BHK-21 cells leads to the formation of at least two types of nsP3-containing complexes, one of which was found in association with the plasma membrane and endosome-like vesicles, while the second was coisolated with cell nuclei. The latter complexes could be solubilized only with the cytoskeleton-destabilizing detergent. Besides viral nsPs, in the mammalian cells, both complexes contained G3BP1 and G3BP2 (which were found in different ratios), YBX1, and HSC70. Rasputin, an insect cell-specific homolog of G3BP1, was found in the nsP3-containing complexes isolated from mosquito cells, which was suggestive of a high conservation of the complexes in the cells of both vertebrate and invertebrate origin. The endosome-and plasma membrane-associated complexes contained a high concentration of double-stranded RNAs (dsRNAs), which is indicative of their function in viral-RNA synthesis. The dsRNA synthesis is likely to efficiently proceed on the plasma membrane, and at least some of the protein-RNA complexes would then be transported into the cytosol in association with the endosome-like vesicular organelles. These findings provide new insight into the mechanism of SINV replication and virus-host cell interactions.The genus Alphavirus in the family Togaviridae contains a number of widely distributed human and animal pathogens. Some of the alphaviruses, including Venezuelan (VEEV), eastern, and western equine encephalitis viruses, constitute a serious public health threat in the United States (53,63,65,66) and cause severe encephalitis in humans and animals that can result in death or neurological sequelae (10,21,27,41). Other family members cause a mild infection, a self-limited febrile illness characterized by fever, skin rashes, and arthritis (21). In spite of differences in their abilities to cause disease, alphaviruses demonstrate strong homology in their encoded proteins and appear to have similar mechanisms of RNA replication (59). Under natural conditions, alphaviruses circulate between mosquito vectors, in which they cause a persistent, life-long infection, and vertebrate hosts, in which the infection is always acute and characterized by a short-term, high-titer viremia that is required for infection of new mosquitoes during blood meals (64). Thus, alphaviruses are capable of replicating in both vertebrate and invertebrate cells and, accordingly, utilize very different intracellular environments for the efficient synthesis of virus-specific RNAs and the production of viral particles.The alphavirus genome is a single-...

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Cold Shock Domain Family Members YB-1 and MSY4 Share Essential Functions during Murine Embryogenesis

Cited by 74 publications

References 27 publications

Unbiased RNA–protein interaction screen by quantitative proteomics

Unbiased RNA–protein interaction screen by quantitative proteomics

Mechanisms of translational repression of the Smcp mRNA in round spermatids

Different Types of nsP3-Containing Protein Complexes in Sindbis Virus-Infected Cells

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