UVM Theses and Dissertations
Format:
Print
Author:
Yang, Qin
Dept./Program:
Microbiology and Molecular Genetics
Year:
2011
Degree:
PhD
Abstract:
Eukaryotic messenger RNA (mRNA) undergoes extensive processing and editing prior to export out of the nucleus and subsequent translation in the cytoplasm. 3' processing is one of the essential steps in the maturation of all messenger RNAs and is a tightly coupled two-step reaction: Endonucleolytic cleavage at the poly(A) site is followed by the addition of a poly(A) tail, except for metazoan histone mRNAs, which are cleaved but not polyadenylated. The recognition of a poly(A) site is coordinated by the sequence elements in the mRNA 3' UTR and associated protein factors. In mammalian cells, three well-studied sequence: elements, AAUAAA, GU-rich and UGUA, are recognized by three multi-subunit factors: cleavage polyadenylation specificity factor, cleavage stimulation factor and cleavage factor Im, respectively. Structural studies of protein-RNA complexes help decipher the mechanisms underlying sequence recognition and shed light on the role of protein factors in poly(A) site selection and 3' processing machinery assembly.
We focused on the interactions between human cleavage factor Im (CFlm) and the UGUA sequence it recognizes, due to both the highly specific nature of this recognition and also the previously implied role of CFlm in alternative polyadenylation, a process that could expand the complexity of gene regulation. CFlm is composed of a small 25 kDa subunit and a larger 59, 68 or 72 kDa subunit. Although the crystal structure of the 25 kDa subunit CFIm25 has been solved, the mechanism for sequence specific recognition of UGUA elements by CFlm complex is yet to be determined. The other protein factor we focused on is poly(A) polymerase (PAP), the enzyme that catalyzes poly(A) tail addition. A structure of PAP in complex with RNA and ATP would further our understanding of the catalytic mechanism of this enzyme.
In this study, we solved the crystal structures of a CFlm25-RNA binary complex and a CFlm25-CFlm68-RNA ternary complex. These structures together with biochemical data demonstrated that 1) CFIm25 is the subunit that is responsible for UGUA recognition. 2) CFIm25 forms a stable dimer in solution and is capable of binding two UGUA elements simultaneously. 3) Two CFlm68 molecules bind to the CFlm25 dimer and facilitate RNA looping between two UGUA elements. 4) CFIm can potentially loop out an entire poly(A) site in between two UGUA and achieve alternative polyadenylation.
These crystal structures not only elucidated the molecular mechanism for sequence specific recognition, but also led to an exciting model for how CFlm may regulate alternative poly(A) site usage. In parallel, we have successfully obtained crystals of a disulfide cross-linked PAP-RNA complex, by implementing a method previously applied to protein-DNA complexes. A structure of a PAP-RNA complex might elucidate the mechanism PAP employs to specifically recognize ATP and uncover the path the RNA follows when bound by the polymerase.
We focused on the interactions between human cleavage factor Im (CFlm) and the UGUA sequence it recognizes, due to both the highly specific nature of this recognition and also the previously implied role of CFlm in alternative polyadenylation, a process that could expand the complexity of gene regulation. CFlm is composed of a small 25 kDa subunit and a larger 59, 68 or 72 kDa subunit. Although the crystal structure of the 25 kDa subunit CFIm25 has been solved, the mechanism for sequence specific recognition of UGUA elements by CFlm complex is yet to be determined. The other protein factor we focused on is poly(A) polymerase (PAP), the enzyme that catalyzes poly(A) tail addition. A structure of PAP in complex with RNA and ATP would further our understanding of the catalytic mechanism of this enzyme.
In this study, we solved the crystal structures of a CFlm25-RNA binary complex and a CFlm25-CFlm68-RNA ternary complex. These structures together with biochemical data demonstrated that 1) CFIm25 is the subunit that is responsible for UGUA recognition. 2) CFIm25 forms a stable dimer in solution and is capable of binding two UGUA elements simultaneously. 3) Two CFlm68 molecules bind to the CFlm25 dimer and facilitate RNA looping between two UGUA elements. 4) CFIm can potentially loop out an entire poly(A) site in between two UGUA and achieve alternative polyadenylation.
These crystal structures not only elucidated the molecular mechanism for sequence specific recognition, but also led to an exciting model for how CFlm may regulate alternative poly(A) site usage. In parallel, we have successfully obtained crystals of a disulfide cross-linked PAP-RNA complex, by implementing a method previously applied to protein-DNA complexes. A structure of a PAP-RNA complex might elucidate the mechanism PAP employs to specifically recognize ATP and uncover the path the RNA follows when bound by the polymerase.