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Pre-mRNA from erythroid enriched bone marrow cells of the rabbit. III. Poly(A)-, oligo(U)- and double stranded
Abstract:
Pre-mRNA from bone marrow of rabbits enriched in erythroid cells was analyzed by T1 and pancreatic RNase treatment and poly(U)- and poly(A)-Sepharose chromatography to contain poly(A)-, oligo(U)- and double stranded sequences. The length of the poly(A)- and oligo(U)-sequences was determined by polyacrylamide gel electrophoresis using poly(A)- and oligo(U)-standards of defined length. Poly(A) from poly(A)+pre-mRNA isolated according to the method of Holmes and Bonner shows a size distribution between 40 and 130 nucleotides with an average of 75 nucleotides. Hot phenol extraction according to Georgiev et al. leads to a smaller size of about 25 nucleotides. The oligo(U)-segment consists of 80% U and is about 25 nucleotides long. Poly(A)+ pre-mRNA of about 12000--16000 nucleotides posseses 1--2 oligo(U)-units and one double strand of about 70 nucleotide pairs. Most (greater than 90%) of the oligo(U)-and the double stranded sequences are localized at least 1700 nucleotides away from the 3'terminus. Double strands were investigated with respect to their reannealing behaviour. The material consists of two types of double strands: 20% which reassociate at a cot/2 cot/2 of 1.3 . 10(-4) represent only one or a few types of double strands, the remaining 80% reassociate at a cot/2 of about 7 . 10(-2) and are more complex. Under hybridization conditions pre-mRNA molecules are able to self-annealation. 10% of the sequences become RNase stable.
Insights
Rabbit erythroid cell pre-mRNA contains poly(A), oligo(U), and double-stranded RNA sequences. These sequences, primarily located away from the 3
Area of Science:
- Molecular Biology
- RNA Biochemistry
- Erythropoiesis
Background:
- Pre-messenger RNA (pre-mRNA) in eukaryotic cells contains various sequence elements crucial for its processing and function.
- Erythroid cells, specialized for oxygen transport, undergo significant gene expression changes during differentiation, making their pre-mRNA a relevant subject for study.
- Understanding the structure and organization of pre-mRNA sequences provides insights into gene regulation and RNA metabolism.
Purpose of the Study:
- To analyze the structural components of pre-mRNA from rabbit erythroid cells.
- To characterize the size, distribution, and properties of poly(A), oligo(U), and double-stranded RNA sequences within this pre-mRNA.
- To investigate the localization and reannealing behavior of these non-coding sequences.
Main Methods:
- Isolation of pre-mRNA from rabbit bone marrow enriched in erythroid cells.
- Enzymatic treatment with T1 and pancreatic RNases.
- Chromatographic separation using poly(U)- and poly(A)-Sepharose.
- Size determination via polyacrylamide gel electrophoresis.
- Analysis of double-strand reannealing kinetics (Cot analysis).
Main Results:
- Pre-mRNA contains poly(A) sequences (average 75 nucleotides, range 40-130 nucleotides), oligo(U) sequences (approx. 25 nucleotides, 80% U), and double-stranded RNA regions (approx. 70 base pairs).
- Oligo(U) and double-stranded sequences are predominantly located far from the 3' terminus ( >1700 nucleotides).
- Double-stranded regions exhibit complex reannealing kinetics, suggesting the presence of multiple sequence types.
Conclusions:
- Rabbit erythroid pre-mRNA possesses distinct structural elements including poly(A), oligo(U), and double-stranded RNA sequences.
- The remote location of oligo(U) and double-stranded sequences from the 3' end suggests potential roles in RNA processing or stability.
- The complexity of double-stranded regions indicates diverse origins or functions within the pre-mRNA molecule.