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Testis/brain RNA-binding protein attaches translationally repressed and transported mRNAs to microtubules
Abstract:
We have previously identified a testicular phosphoprotein that binds to highly conserved sequences (Y and H elements) in the 3' untranslated regions (UTRs) of testicular mRNAs and suppresses in vitro translation of mRNA constructs that contain these sequences. This protein, testis/brain RNA-binding protein (TB-RBP) also is abundant in brain and binds to brain mRNAs whose 3' UTRs contain similar sequences. Here we show that TB-RBP binds specific mRNAs to microtubules (MTs) in vitro. When TB-RBP is added to MTs reassembled from either crude brain extracts or from purified tubulin, most of the TB-RBP binds to MTs. The association of TB-RBP with MTs requires the assembly of MTs and is diminished by colcemid, cytochalasin D, and high levels of salt. Transcripts from the 3' UTRs of three mRNAs that contain the conserved sequence elements (transcripts for protamine 2, tau protein, and myelin basic protein) are linked by TB-RBP to MTs, whereas transcripts that lack the conserved sequences do not bind TB-RBP. We conclude that TB-RBP serves as an attachment protein for the MT association of specific mRNAs. Considering its ability to arrest translation in vitro, we propose that TB-RBP functions in the storage and transportation of mRNAs to specific intracellular sites where they are translated.
Insights
Testis/brain RNA-binding protein (TB-RBP) attaches specific mRNAs to microtubules (MTs). This suggests TB-RBP stores and transports mRNAs for localized translation.
Area of Science:
- Molecular Biology
- Neuroscience
- Cell Biology
Background:
- A testicular phosphoprotein, testis/brain RNA-binding protein (TB-RBP), binds conserved sequences in mRNA 3' UTRs.
- TB-RBP suppresses in vitro translation and is also found in the brain, binding brain mRNAs with similar sequences.
Purpose of the Study:
- To investigate the interaction of TB-RBP with microtubules (MTs).
- To determine if TB-RBP mediates mRNA binding to MTs.
Main Methods:
- In vitro binding assays using reassembled MTs from brain extracts or purified tubulin.
- Assessing TB-RBP association with MTs under various conditions (colcemid, cytochalasin D, salt).
- Analyzing the binding of specific mRNA transcripts (protamine 2, tau, myelin basic protein) to MTs via TB-RBP.
Main Results:
- TB-RBP binds specifically to assembled MTs in vitro.
- MT assembly is required for TB-RBP association, which is sensitive to colcemid, cytochalasin D, and high salt concentrations.
- TB-RBP links specific mRNAs containing conserved sequence elements to MTs, while mRNAs lacking these sequences do not bind.
Conclusions:
- TB-RBP acts as an attachment protein, mediating the MT association of specific mRNAs.
- TB-RBP may function in mRNA storage and transport to intracellular sites for localized translation, consistent with its in vitro translation-inhibiting activity.