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U11/U12-65K is a spliceosomal protein encoded by the RNPC3 gene in humans. It is a component of the U11/U12 di-snRNP and one of the proteins found in the U12-dependent spliceosome, but not in the U2-dependent spliceosome.
IntroductionMost eukaryotic genomes harbor two types of spliceosomal introns, called U2-type and U12-type introns, which are excised by two different spliceosomes[1]. U12-type introns are rare, and only present a small fraction of introns in any given eukaryotic genome. The U12-dependent spliceosome, also called the minor spliceosome, is responsible for the removal of these rare introns. Both spliceosomes consist of five small nuclear ribonucleoprotein particles (snRNPs), which are U1, U2, U4, U5 and U6 for the U2-dependent spliceosome and U11, U12, U4atac, U5 and U6atac for the U12-dependent spliceosome. The assembly pathways of the two spliceosomes are similar, but the initial intron recognition occurs differently. In the case of U2-type introns, the 5' splice site (5'ss) is recognized by the U1 snRNP, while the recognition of the branch-point sequence (BPS), the polypyrimidine tract (PPT) and the 3' splice site (3'ss) is carried out by the protein factors SF1, U2AF65 and U2AF35, respectively. Subsequently, SF1 is replaced at the BPS by U2 snRNP. In contrast, the U12-type 5'ss and BPS are recognized by the U11/U12 di-snRNP, which binds the intron as a preformed complex. The protein composition of the two spliceosomes is similar, and so far only seven proteins specific for the minor spliceosome have been identified[2][3][4]. All seven proteins (called 65K, 59K, 48K, 35K, 31K, 25K and 20K) are components of the U11/U12 di-snRNP[4]. The U11/U12-65K protein forms part of a molecular bridge that connects the U11 and U12 snRNPs[5], and is important for the stability of the U11/U12 di-snRNP and splicing of U12-type introns[6][7][8]. It binds to U12 snRNA and U11-59K[5], a protein component of the U11 snRNP. The U11-59K protein further interacts with U11-48K, a protein involved in the recognition of the U12-type 5' splice site[9]. Mutations in the gene encoding the U11/U12-65K protein (RNPC3) were recently shown to cause isolated growth hormone deficiency (IGHD) and pituitary hypoplasia[6]. StructureDomain structureThe human U11/U12-65K protein contains two RNA recognition motifs (RRM), one close to the N-terminus (residues 27–102) and another one near the C-terminus (residues 420–503), as well as a proline-rich region (residues 196–233) located between the two RRMs. The C-terminal RRM exhibits higher conservation, and is likely homologous to the N-terminal RRMs of the U1A and U2B" proteins[5], which are components of the U1 and U2 snRNPs, respectively. The C-terminal RRM binds specifically to stem-loop III of U12 snRNA, whereas the N-terminal half of the protein (residues 1–257), which includes the second RRM and the proline-rich region, interacts with the U11-59K protein[5]. Crystal structure of the C-terminal RRM of U11/U12-65KThe structure of the C-terminal RNA recognition motif and an associated N-terminal extension (residues 380–517) of human U11/U12-65K has been determined by X-ray crystallography at 2.5 Å resolution[10] (, coloring by secondary structure). The protein was crystallized in complex with an RNA oligonucleotide containing the loop and part of the stem of U12 snRNA stem-loop III; however, the oligonucleotide was not visible in the electron density. Residues 387-506 of the protein could be traced from the electron density map. The structure was solved by the molecular replacement method, using coordinates from the structure of the closely related U1A N-terminal RRM (PDB ID: 1urn). In the 65K C-terminal RRM structure, residues 417–501 adopt a typical RRM fold, with an antiparallel four-stranded β-sheet packed against two α-helices. In addition to these canonical elements, the 65K C-terminal RRM contains between the α1 helix and the β2 strand and a in the loop connecting β2 and α2. Interestingly, as shown by electrophoretic mobility shift assays (EMSA), the core RRM fold of U11/U12-65K alone fails to bind to U12 snRNA. The core fold is stabilized by an N-terminal expansion comprising residues 380–417, which are required for RNA binding. folds into two α-helices, a 310-helix and a long loop that connects the expansion to the β1 strand. Most RRMs interact with RNA through their β-sheet surface, which contains two highly conserved sequence motifs, called RNP1 and RNP2, located in the central β3 and β1 strands, respectively[11][12]. In many cases, additional elements such as the loops connecting various secondary structure elements, or the N- or C-terminal extensions outside the core RRM fold are also involved in RNA binding. The structural basis of RNA binding of the U11/U12-65K C-terminal RRM appears to be similar to that of canonical RRMs, such as the U1A N-terminal RRM. Because of the absence of RNA in the 65K structure, direct structural information on RNA–protein contacts is not available. However, based on sequence alignment, Netter et al. identified several residues analogous to those involved in RNA binding in the U1A protein, mutated these residues and tested their effect on RNA binding using gel-shift assays[10]. When the was mutated to alanine or phenylalanine, RNA binding was abolished. This residue corresponds to the Tyr13 residue of the U1A N-terminal RRM and is located on the β-sheet surface in the β1 strand. Thus, similar to U1A N-terminal RRM and other canonical RRMs, the β-sheet surface plays an important role in RNA binding in the U11/U12-65K C-terminal RRM. In U1A, residues in the loop connecting β2 and β3 are also involved in RNA binding by interacting with the bases or the backbone of the RNA. Mutating to glutamine in the corresponding loop in the 65K C-terminal RRM led to a large reduction in binding affinity in the case of Arg464 and complete abolishment of RNA binding in the case of Lys466. These residues correspond to residues Lys50 and Arg52 in the U1A N-terminal RRM. In conclusion, both the β-sheet surface and the β2–β3 seem to contribute to RNA binding in the 65K C-terminal RRM. In contrast, mutating potential RNA-contacting residues (Arg411, Val409) in the N-terminal expansion had only a moderate effect on RNA affinity. However, the N-terminal expansion is clearly essential for RNA binding, since a protein construct lacking the N-terminal expansion had no RNA-binding activity. Thermal denaturation assays suggested that the role of the N-terminal expansion is to stabilize the core RRM fold, which alone is unable to bind RNA. References
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