Structural basis for the sequence-specific RNA-recognition mechanism of human CUG-BP1 RRM3

The CUG-binding protein 1 (CUG-BP1) is a member of the CUG-BP1 and ETR-like factors (CELF) family or the Bruno-like family and is involved in the control of splicing, translation and mRNA degradation. Several target RNA sequences of CUG-BP1 have been predicted, such as the CUG triplet repeat, the GU-rich sequences and the AU-rich element of nuclear pre-mRNAs and/or cytoplasmic mRNA. CUG-BP1 has three RNA-recognition motifs (RRMs), among which the third RRM (RRM3) can bind to the target RNAs on its own. In this study, we solved the solution structure of the CUG-BP1 RRM3 by hetero-nuclear NMR spectroscopy. The CUG-BP1 RRM3 exhibited a noncanonical RRM fold, with the four-stranded b-sheet surface tightly associated with the N-terminal extension. Furthermore, we determined the solution structure of the CUG-BP1 RRM3 in the complex with (UG)3 RNA, and discovered that the UGU trinucleotide is specifically recognized through extensive stacking interactions and hydrogen bonds within the pocket formed by the b-sheet surface and the N-terminal extension. This study revealed the unique mechanism that enables the CUG-BP1 RRM3 to discriminate the short RNA segment from other sequences, thus providing the molecular basis for the comprehension of the role of the RRM3s in the CELF/Bruno-like family

An Arabidopsis SBP-domain fragment with a disrupted C-terminal zinc-binding site retains its tertiary structure

AbstractSQUAMOSA promoter-binding proteins (SBPs) form a major family of plant-specific transcription factors, mainly related to flower development. SBPs share a highly conserved DNA-binding domain of ∼80 amino acids (SBP domain), which contains two non-interleaved zinc-binding sites formed by eight conserved Cys or His residues. In the present study, an Arabidopsis SPL12 SBP-domain fragment that lacks a Cys residue involved in the C-terminal zinc-binding pocket was found to retain a folded structure, even though only a single Zn2+ ion binds to the fragment. Solution structure of this fragment determined by NMR is very similar to the previously determined structures of the full SBP domains of Arabidopsis SPL4 and SPL7. Considering the previous observations that chelating all the Zn2+ ions of SBPs resulted in the complete unfolding of the structure and that a mutation of the Cys residue equivalent to that described above impaired the DNA-binding activity, we propose that the Zn2+ ion at the N-terminal site is necessary to maintain the overall tertiary structure, while the Zn2+ ion at the C-terminal site is necessary for the DNA binding, mainly by guiding the basic C-terminal loop to correctly fit into the DNA groove

Structural basis for the dual RNA-recognition modes of human Tra2-beta RRM

Human Transformer2-beta (hTra2-beta) is an important member of the serine/arginine-rich protein family, and contains one RNA recognition motif (RRM). It controls the alternative splicing of several pre-mRNAs, including those of the calcitonin/calcitonin gene-related peptide (CGRP), the survival motor neuron 1 (SMN1) protein and the tau protein. Accordingly, the RRM of hTra2-beta specifically binds to two types of RNA sequences [the CAA and (GAA)2 sequences]. We determined the solution structure of the hTra2-beta RRM (spanning residues Asn110–Thr201), which not only has a canonical RRM fold, but also an unusual alignment of the aromatic amino acids on the beta-sheet surface. We then solved the complex structure of the hTra2-beta RRM with the (GAA)2 sequence, and found that the AGAA tetra-nucleotide was specifically recognized through hydrogen-bond formation with several amino acids on the N- and C-terminal extensions, as well as stacking interactions mediated by the unusually aligned aromatic rings on the beta-sheet surface. Further NMR experiments revealed that the hTra2-beta RRM recognizes the CAA sequence when it is integrated in the stem-loop structure. This study indicates that the hTra2-beta RRM recognizes two types of RNA sequences in different RNA binding modes

The RRM domain of poly(A)-specific ribonuclease has a noncanonical binding site for mRNA cap analog recognition

The degradation of the poly(A) tail is crucial for posttranscriptional gene regulation and for quality control of mRNA. Poly(A)-specific ribonuclease (PARN) is one of the major mammalian 3′ specific exo-ribonucleases involved in the degradation of the mRNA poly(A) tail, and it is also involved in the regulation of translation in early embryonic development. The interaction between PARN and the m7GpppG cap of mRNA plays a key role in stimulating the rate of deadenylation. Here we report the solution structures of the cap-binding domain of mouse PARN with and without the m7GpppG cap analog. The structure of the cap-binding domain adopts the RNA recognition motif (RRM) with a characteristic α-helical extension at its C-terminus, which covers the β-sheet surface (hereafter referred to as PARN RRM). In the complex structure of PARN RRM with the cap analog, the base of the N7-methyl guanosine (m7G) of the cap analog stacks with the solvent-exposed aromatic side chain of the distinctive tryptophan residue 468, located at the C-terminal end of the second β-strand. These unique structural features in PARN RRM reveal a novel cap-binding mode, which is distinct from the nucleotide recognition mode of the canonical RRM domains

Cell-Free Protein Synthesis Using S30 Extracts from <i>Escherichia coli</i> RFzero Strains for Efficient Incorporation of Non-Natural Amino Acids into Proteins

Cell-free protein synthesis is useful for synthesizing difficult targets. The site-specific incorporation of non-natural amino acids into proteins is a powerful protein engineering method. In this study, we optimized the protocol for cell extract preparation from the Escherichia coli strain RFzero-iy, which is engineered to lack release factor 1 (RF-1). The BL21(DE3)-based RFzero-iy strain exhibited quite high cell-free protein productivity, and thus we established the protocols for its cell culture and extract preparation. In the presence of 3-iodo-l-tyrosine (IY), cell-free protein synthesis using the RFzero-iy-based S30 extract translated the UAG codon to IY at various sites with a high translation efficiency of &gt;90%. In the absence of IY, the RFzero-iy-based cell-free system did not translate UAG to any amino acid, leaving UAG unassigned. Actually, UAG was readily reassigned to various non-natural amino acids, by supplementing them with their specific aminoacyl-tRNA synthetase variants (and their specific tRNAs) into the system. The high incorporation rate of our RFzero-iy-based cell-free system enables the incorporation of a variety of non-natural amino acids into multiple sites of proteins. The present strategy to create the RFzero strain is rapid, and thus promising for RF-1 deletions of various E. coli strains genomically engineered for specific requirements

Expression, purification, crystallization and preliminary X-ray diffraction analysis of galactokinase from Pyrococcus horikoshii

Galactokinase from P. horikoshii has been crystallized in both the apo form and as a ternary complex with α-d-galactose and an ATP analogue. The crystals were characterized by X-ray diffraction. The kinetic parameters of the enzyme were determined