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The polyribosomal protein bound to the 3' end of histone mRNA can function in histone pre-mRNA processing
Z Dominski1, J Sumerel, R J Hanson
1Department of Biochemistry, University of North Carolina, Chapel Hill 27599, USA.
Abstract:
Cell cycle-regulated histone mRNAs end in a conserved 26-nt sequence that can form a stem-loop with a six-base stem and a four-base loop. The 3' end of histone mRNA has distinct functions in the nucleus and in the cytoplasm. In the nucleus it functions in pre-mRNA processing and transport, whereas in the cytoplasm it functions in translation and regulation of histone mRNA stability. The stem-loop binding protein (SLBP), present in both nuclei and polyribosomes, is likely the trans-acting factor that binds to the 3' end of mature histone mRNA and mediates its function. A nuclear extract that efficiently processes histone pre-mRNA was prepared from mouse myeloma cells. The factor(s) that bind to the 3' end of histone mRNA can be depleted from this extract using a biotinylated oligonucleotide containing the conserved stem-loop sequence. Using this depleted extract which is deficient in histone pre-mRNA processing, we show that SLBP found in polyribosomes can restore processing, suggesting that SLBP associates with histone pre-mRNA in the nucleus, participates in processing, and then accompanies the mature mRNA to the cytoplasm.
Insights
Stem-loop binding protein (SLBP) is crucial for histone mRNA processing and function. SLBP binds to the conserved 3' end stem-loop of histone mRNA, facilitating its transport and translation in both nucleus and cytoplasm.
Area of Science:
- Molecular Biology
- Gene Regulation
- Cell Biology
Background:
- Histone mRNAs possess a conserved 26-nucleotide 3' end sequence capable of forming a stem-loop structure.
- This 3' end plays distinct roles in nuclear pre-mRNA processing and cytoplasmic mRNA translation and stability.
- Stem-loop binding protein (SLBP) is implicated as a key trans-acting factor interacting with this structure.
Purpose of the Study:
- To investigate the role of SLBP in histone mRNA processing and function.
- To determine if SLBP mediates the distinct nuclear and cytoplasmic functions of the histone mRNA 3' end.
- To elucidate the mechanism by which SLBP associates with histone pre-mRNA and mature mRNA.
Main Methods:
- Preparation of a nuclear extract from mouse myeloma cells with efficient histone pre-mRNA processing activity.
- Depletion of SLBP from the nuclear extract using a biotinylated oligonucleotide targeting the conserved stem-loop sequence.
- Restoration of histone pre-mRNA processing in the depleted extract by adding back SLBP from polyribosomes.
Main Results:
- A nuclear extract capable of processing histone pre-mRNA was successfully prepared.
- Depletion of SLBP-binding factors from the extract abolished histone pre-mRNA processing.
- Reintroduction of SLBP from polyribosomes restored the processing activity of the depleted extract.
Conclusions:
- SLBP binds to the conserved 3' stem-loop sequence of histone mRNA.
- SLBP is essential for histone pre-mRNA processing in the nucleus.
- SLBP accompanies mature histone mRNA to the cytoplasm, participating in its post-transcriptional regulation.