Purification of Messenger Ribonucleoprotein Particles via a Tagged Nascent Polypeptide.
The cytoplasmic fates of mRNAs are influenced by interactions between RNA-binding proteins and cis regulatory motifs. In the cytoplasm, mRNAs are present as messenger ribonucleoprotein particles, which include not only proteins that bind directly to the mRNA, but also additional proteins that are re...
| Main Authors: | , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2016-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4726818?pdf=render |
| _version_ | 1811205659003191296 |
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| author | Diana P Inchaustegui Gil Christine Clayton |
| author_facet | Diana P Inchaustegui Gil Christine Clayton |
| author_sort | Diana P Inchaustegui Gil |
| collection | DOAJ |
| description | The cytoplasmic fates of mRNAs are influenced by interactions between RNA-binding proteins and cis regulatory motifs. In the cytoplasm, mRNAs are present as messenger ribonucleoprotein particles, which include not only proteins that bind directly to the mRNA, but also additional proteins that are recruited via protein-protein interactions. Many labs have sought to purify such particles from cells, with limited success. We here describe a simple two-step procedure to purify actively translated mRNAs, with their associated proteins, from polysomes. We use a reporter mRNA that encodes a protein with three streptavidin binding peptides at the N-terminus. The polysomal reporter mRNA, with associated proteins, is purified via binding to a streptavidin matrix. The method takes four days, and can be applied in any cell that can be genetically manipulated. Using Trypanosoma brucei as a model system, we routinely purified 8% of the input reporter mRNA, with roughly 22-fold enrichment relative to un-tagged mRNAs, a final reporter-mRNA:total-mRNA ratio of about 1:10, and a protein purification factor of slightly over 1000-fold. Although the overall reporter mRNP composition is masked by the presence of proteins that are associated with many polysomal mRNAs, our method can be used to detect association of an RNA-binding protein that binds to specifically to a reporter mRNA. |
| first_indexed | 2024-04-12T03:35:49Z |
| format | Article |
| id | doaj.art-ad435e85c61546b6b8f21df960438dd6 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-12T03:35:49Z |
| publishDate | 2016-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-ad435e85c61546b6b8f21df960438dd62022-12-22T03:49:26ZengPublic Library of Science (PLoS)PLoS ONE1932-62032016-01-01111e014813110.1371/journal.pone.0148131Purification of Messenger Ribonucleoprotein Particles via a Tagged Nascent Polypeptide.Diana P Inchaustegui GilChristine ClaytonThe cytoplasmic fates of mRNAs are influenced by interactions between RNA-binding proteins and cis regulatory motifs. In the cytoplasm, mRNAs are present as messenger ribonucleoprotein particles, which include not only proteins that bind directly to the mRNA, but also additional proteins that are recruited via protein-protein interactions. Many labs have sought to purify such particles from cells, with limited success. We here describe a simple two-step procedure to purify actively translated mRNAs, with their associated proteins, from polysomes. We use a reporter mRNA that encodes a protein with three streptavidin binding peptides at the N-terminus. The polysomal reporter mRNA, with associated proteins, is purified via binding to a streptavidin matrix. The method takes four days, and can be applied in any cell that can be genetically manipulated. Using Trypanosoma brucei as a model system, we routinely purified 8% of the input reporter mRNA, with roughly 22-fold enrichment relative to un-tagged mRNAs, a final reporter-mRNA:total-mRNA ratio of about 1:10, and a protein purification factor of slightly over 1000-fold. Although the overall reporter mRNP composition is masked by the presence of proteins that are associated with many polysomal mRNAs, our method can be used to detect association of an RNA-binding protein that binds to specifically to a reporter mRNA.http://europepmc.org/articles/PMC4726818?pdf=render |
| spellingShingle | Diana P Inchaustegui Gil Christine Clayton Purification of Messenger Ribonucleoprotein Particles via a Tagged Nascent Polypeptide. PLoS ONE |
| title | Purification of Messenger Ribonucleoprotein Particles via a Tagged Nascent Polypeptide. |
| title_full | Purification of Messenger Ribonucleoprotein Particles via a Tagged Nascent Polypeptide. |
| title_fullStr | Purification of Messenger Ribonucleoprotein Particles via a Tagged Nascent Polypeptide. |
| title_full_unstemmed | Purification of Messenger Ribonucleoprotein Particles via a Tagged Nascent Polypeptide. |
| title_short | Purification of Messenger Ribonucleoprotein Particles via a Tagged Nascent Polypeptide. |
| title_sort | purification of messenger ribonucleoprotein particles via a tagged nascent polypeptide |
| url | http://europepmc.org/articles/PMC4726818?pdf=render |
| work_keys_str_mv | AT dianapinchausteguigil purificationofmessengerribonucleoproteinparticlesviaataggednascentpolypeptide AT christineclayton purificationofmessengerribonucleoproteinparticlesviaataggednascentpolypeptide |