The elongation factor eEF3 (Yef3) interacts with mRNA in a translation independent manner

Samra, Nitzan; Atir-Lande, Avigail; Pnueli, Lilach; Arava, Yoav

doi:10.1186/s12867-015-0045-5

Cited by 10 publications

(13 citation statements)

References 50 publications

(64 reference statements)

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“…Another optimized in vivo system, RBP purification and identification (RaPID), uses a novel fusion protein, MS2cp-GFP-SBP, which enables both affinity purification of RNA–protein complexes using streptavidin-conjugated beads due to interaction with SBP and visualization of intracellular localization of target mRNAs bearing the MS2 aptamer using microscopy due to a fluorescence reporter (MS2cp-GFP) (44,77). Specific and strong binding of MS2cp to the MS2 loops and of SBP to streptavidin as well as formaldehyde crosslinking allows for stringent washing conditions, which prevent isolation of nonspecific RNA–protein interactions.…”

Section: Purification Of In Vitro- and In Vivo-assembled Rna–protein mentioning

confidence: 99%

“…Another advantage of RaPID analysis is simple and specific elution of RNP complexes by competition with biotin. Additionally, to avoid MS2cp aggregation following high expression levels, genes encoding fusion proteins were placed under the control of an inducible promoter, which allow tightly regulated stable expression in both yeast and mammalian cells (44,77). …”

Section: Purification Of In Vitro- and In Vivo-assembled Rna–protein mentioning

confidence: 99%

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Identifying proteins that bind to specific RNAs - focus on simple repeat expansion diseases

Jazurek¹,

Ciesiolka²,

Staręga-Rosłan³

et al. 2016

Nucleic Acids Res

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RNA–protein complexes play a central role in the regulation of fundamental cellular processes, such as mRNA splicing, localization, translation and degradation. The misregulation of these interactions can cause a variety of human diseases, including cancer and neurodegenerative disorders. Recently, many strategies have been developed to comprehensively analyze these complex and highly dynamic RNA–protein networks. Extensive efforts have been made to purify in vivo-assembled RNA–protein complexes. In this review, we focused on commonly used RNA-centric approaches that involve mass spectrometry, which are powerful tools for identifying proteins bound to a given RNA. We present various RNA capture strategies that primarily depend on whether the RNA of interest is modified. Moreover, we briefly discuss the advantages and limitations of in vitro and in vivo approaches. Furthermore, we describe recent advances in quantitative proteomics as well as the methods that are most commonly used to validate robust mass spectrometry data. Finally, we present approaches that have successfully identified expanded repeat-binding proteins, which present abnormal RNA–protein interactions that result in the development of many neurological diseases.

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Section: Purification Of In Vitro- and In Vivo-assembled Rna–protein mentioning

confidence: 99%

Section: Purification Of In Vitro- and In Vivo-assembled Rna–protein mentioning

confidence: 99%

Identifying proteins that bind to specific RNAs - focus on simple repeat expansion diseases

Jazurek¹,

Ciesiolka²,

Staręga-Rosłan³

et al. 2016

Nucleic Acids Res

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“…However, the insertion of a long sequence of MS2 loops may cause changes in the processing and structure of the RNA; it might destabilize the transcript or change the repertoire of RBPs bound to it. In our experience, 12 loops are sufficient to get an efficient elution of MS2-tagged RNA 20 . In this respect, we note that we usually observed additional shorter transcripts in our northern hybridizations.…”

Section: Discussionmentioning

confidence: 93%

“…In this respect, we note that we usually observed additional shorter transcripts in our northern hybridizations. Northern analyses with different probes revealed that these shorter forms included the MS2L and part of the 3' UTR 20 , indicative of premature transcription termination. Such cases are likely to reduce the efficiency of the isolation of proteins associated with the 3' UTR.…”

Section: Discussionmentioning

confidence: 99%

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Novel RNA-Binding Proteins Isolation by the RaPID Methodology

Samra

Arava

2016

JoVE

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RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

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