A novel occluded RNA recognition motif in Prp24 unwinds the U6 RNA internal stem loop

The essential splicing factor Prp24 contains four RNA Recognition Motif (RRM) domains, and functions to anneal U6 and U4 RNAs during spliceosome assembly. Here, we report the structure and characterization of the C-terminal RRM4. This domain adopts a novel non-canonical RRM fold with two additional flanking α-helices that occlude its β-sheet face, forming an occluded RRM (oRRM) domain. The flanking helices form a large electropositive surface. oRRM4 binds to and unwinds the U6 internal stem loop (U6 ISL), a stable helix that must be unwound during U4/U6 assembly. NMR data indicate that the process starts with the terminal base pairs of the helix and proceeds toward the loop. We propose a mechanistic and structural model of Prp24′s annealing activity in which oRRM4 functions to destabilize the U6 ISL during U4/U6 assembly

Crystal Structure of the RNA Recognition Motif of Yeast Translation Initiation Factor eIF3b Reveals Differences to Human eIF3b

BACKGROUND: The multi-subunit eukaryotic initiation factor3 (eIF3) plays a central role in the initiation step of protein synthesis in eukaryotes. One of its large subunits, eIF3b, serves as a scaffold within eIF3 as it interacts with several other subunits. It harbors an RNA Recognition Motif (RRM), which is shown to be a non-canonical RRM in human as it is not capable to interact with oligonucleotides, but rather interacts with eIF3j, a sub-stoichiometric subunit of eIF3. PRINCIPAL FINDING: We have analyzed the high-resolution crystal structure of the eIF3b RRM domain from yeast. It exhibits the same fold as its human ortholog, with similar charge distribution on the surface interacting with the eIF3j in human. Thermodynamic analysis of the interaction between yeast eIF3b-RRM and eIF3j revealed the same range of enthalpy change and dissociation constant as for the human proteins, providing another line of evidence for the same mode of interaction between eIF3b and eIF3j in both organisms. However, analysis of the surface charge distribution of the putative RNA-binding β-sheet suggested that in contrast to its human ortholog, it potentially could bind oligonucleotides. Three-dimensional positioning of the so called "RNP1" motif in this domain is similar to other canonical RRMs, suggesting that this domain might indeed be a canonical RRM, conferring oligonucleotide binding capability to eIF3 in yeast. Interaction studies with yeast total RNA extract confirmed the proposed RNA binding activity of yeast eIF3b-RRM. CONCLUSION: We showed that yeast eIF3b-RRM interacts with eIF3j in a manner similar to its human ortholog. However, it shows similarities in the oligonucleotide binding surface to canonical RRMs and interacts with yeast total RNA. The proposed RNA binding activity of eIF3b-RRM may help eIF3 to either bind to the ribosome or recruit the mRNA to the 43S pre-initiation complex

Modular protein-RNA interactions regulating mRNA metabolism: a role for NMR

Here we review the role played by transient interactions between multi-functional proteins and their RNA targets in the regulation of mRNA metabolism, and we describe the important function of NMR spectroscopy in the study of these systems. We place emphasis on a general approach for the study of different features of modular multi-domain recognition that uses well-established NMR techniques and that has provided important advances in the general understanding of post-transcriptional regulation

Structure and functional implications of a complex containing a segment of U6 RNA bound by a domain of Prp24

U6 RNA plays a critical role in pre-mRNA splicing. Assembly of U6 into the spliceosome requires a significant structural rearrangement and base-pairing with U4 RNA. In the yeast Saccharomyces cerevisiae, this process requires the essential splicing factor Prp24. We present the characterization and structure of a complex containing one of Prp24's four RNA recognition motif (RRM) domains, RRM2, and a fragment of U6 RNA. NMR methods were used to identify the preferred U6 binding sequence of RRM2 (5′-GAGA-3′), measure the affinity of the interaction, and solve the structure of RRM2 bound to the hexaribonucleotide AGAGAU. Interdomain contacts observed between RRM2 and RRM3 in a crystal structure of the free protein are not detectable in solution. A structural model of RRM1 and RRM2 bound to a longer segment of U6 RNA is presented, and a partial mechanism for Prp24's annealing activity is proposed