Crystallization of auto-regulatory A-rich repeats found in the 5′-UTR of human PABP mRNA

Abstract

Eukaryotic poly(A)-binding protein (PABP) binds not only to the 3' poly(A) tail of most mRNAs, but also to the 5'-untranslated region (UTR). The latter form of binding leads to auto-regulation of PABP expression. The 5'-UTR sequence contains A-rich repeats different from that in 3'-UTR. The role of these A-rich repeats in auto-regulation, however, remains unknown. To confirm that the 5'-UTR sequence has a specific structure before or after PABP binding, and to determine the function of such structure, several RNA/DNA fragments containing A-rich repeats were examined by crystallization, fluorescence microscopy and X-ray diffraction. Crystals were obtained for several of these fragments. In particular, single crystals were obtained for the DNA fragment with four repeats, suggesting that such fragment is folded into a regular structure through A:A interactions.

Structure of the Parallel Duplex of Poly(A) RNA: Evaluation of a 50 Year‐Old Prediction

“…This structure shows that (rA) 11 forms a parallel doublestranded helix ( Figure 1) with symmetric base pairs formed by two adenine nucleotides (Figure 1 A). The A-A base pairs are "N7-amino-symmetric" where N7 atom of one adenine forms a hydrogen bond with the amino group of the opposite base.…”

“…Subsequently, Pattabiraman [10] showed that formation of a parallel, right-handed nucleic acid duplex is thermodynamically favorable. Kikuchi et al [11] were able to crystallize A-rich fragments of RNA and DNA. Recently, Chakraborty et al, [12] using NMR spectroscopy and thermal denaturation studies, showed that poly(dA) can form a parallel homo duplex in acidic solution with low salt concentration.…”

“…Despite time and effort, the detailed atomic structure of the duplex of poly(rA) has remained elusive. Herein, we obtained highly ordered crystals of (rA) 11 and determined its structure to 1 resolution. Thermal denaturation studies were used to study the stability of the poly(rA) duplex in solution and elucidate the conditions for its formation.…”

“…Crystallization of (rA) 11 required the presence of a fragment of poly(A)-binding protein (PABP) containing domains RRM1-2. The binding of (rA) 11 to the protein most likely reduces the rate of association of the RNA strands to allow the formation of well-ordered crystals.…”

“…Crystallization of (rA) 11 required the presence of a fragment of poly(A)-binding protein (PABP) containing domains RRM1-2. The binding of (rA) 11 to the protein most likely reduces the rate of association of the RNA strands to allow the formation of well-ordered crystals. High-quality data were collected from multiple single crystals and initial phases were determined using ab initio direct methods employing the multi-CPU version of the program ShelxD.…”