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A polyadenylation factor subunit is the human homologue of the Drosophila suppressor of forked protein
1Department of Biological Sciences, Columbia University, New York, New York 10027.
Abstract:
Polyadenylation of messenger RNA precursors is a complex process that requires multiple protein factors (for reviews, see refs 1, 2). Cleavage stimulation factor (CstF) is one of these, functioning together with cleavage-polyadenylation specificity factor, two cleavage factors, and poly(A)+ polymerase. CstF is composed of three subunits of M(r) 77, 64 and 50K. The 64K and 50K subunits contain, respectively, an RNP-type RNA-binding domain that contacts the pre-mRNA and transducin repeats characteristic of G-protein beta-subunits. Here we report the cloning and characterization of the 77K subunit of human CstF (referred to as 77K). We show that the 77K subunit is required for formation of active CstF and bridges the 64K and 50K subunits. Sequence analyses indicate that the 77K subunit is the homologue of the protein encoded by the Drosophila melanogaster suppressor of forked (su(f)) gene. Mutations in su(f) can enhance or suppress the effects of transposable element insertions, and our data indicate that this is due to changes in polyadenylation. Both the 77K subunit and the su(f) protein share homology with Saccharomyces cerevisiae RNA14, previously shown to be involved in mRNA metabolism. Our results thus also indicate that components of the complex polyadenylation machinery are conserved from yeast to man.
Insights
The 77K subunit is essential for forming active human cleavage stimulation factor (CstF), a key protein complex in messenger RNA processing. This finding reveals conserved mechanisms in polyadenylation from yeast to humans.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Messenger RNA (mRNA) polyadenylation is a crucial post-transcriptional modification involving multiple protein factors.
- Cleavage stimulation factor (CstF) is a key component of the polyadenylation machinery, interacting with other factors like cleavage-polyadenylation specificity factor and poly(A)+ polymerase.
- CstF comprises three subunits: 77K, 64K, and 50K, with the 64K and 50K subunits possessing RNA-binding and G-protein beta-subunit-like domains, respectively.
Purpose of the Study:
- To clone and characterize the 77K subunit of human CstF.
- To elucidate the role of the 77K subunit in the assembly and function of the CstF complex.
- To investigate the evolutionary conservation of the 77K subunit and its role in polyadenylation.
Main Methods:
- Cloning and characterization of the 77K subunit of human CstF.
- Functional assays to determine the requirement of the 77K subunit for CstF activity.
- Sequence analysis to identify homologous proteins in other species.
- Comparative genomics to assess evolutionary conservation.
Main Results:
- The 77K subunit is indispensable for the formation of an active CstF complex.
- The 77K subunit acts as a bridge, connecting the 64K and 50K subunits within CstF.
- Sequence homology identifies the 77K subunit as the human homolog of the Drosophila melanogaster suppressor of forked (su(f)) gene product.
- Mutations in su(f) affect polyadenylation, influencing transposable element insertion effects.
- Homology was also found with Saccharomyces cerevisiae RNA14, indicating conserved roles in mRNA metabolism.
Conclusions:
- The 77K subunit is a critical structural and functional component of human CstF.
- The study provides evidence for conserved mechanisms in mRNA polyadenylation across species, from yeast to humans.
- The findings link the su(f) gene in Drosophila to polyadenylation regulation and highlight conserved components of this essential cellular process.