Fmoc amino acid fluorides, recently shown to be a new class of rapid-acting acylating agents in peptide synthesis are well suited for the solid-phase synthesis of medium-sized peptides such as , magainin-II-amide, and h-CRF. The most important advantage of these reagents is their high reactivity in the coupling of sterically hindered amino acid residues, such as a-aminoisobutyric acid (Aib), results which are at least partly due to the small size of the fluoride leaving group. Both h-(Aib32-35)-CRF(l-41), bearing four consecutive Aib-residues, and alamethicin acid, neither previously accessible by solid-phase synthesis, were successfully synthesized via acid fluorides using unusually short coupling times. In contrast, attempted syntheses via UNCA's and PyBroP activation, both reported to be well suited for sterically hindered systems, failed to give the desired peptides. These remarkable differences prompted a more detailed comparison of the acid fluorides with symmetric anhydrides, UNCA's, and the PyBroP activation technique. Side products formed during the acylation of hindered amino components by Fmoc-Aib-NCA were identified and their formation rationalized. These side products could have their origin in the demonstrated instability of Fmoc-NCA's in the presence of tertiary bases or in a diversion of the position of attack on the NCA from the more hindered to the less-hindered carbonyl function by a bulky nucleophile. Clearly caution is required when such bases are employed to enhance coupling rates for hindered systems.

The yeast suppressor of myosin 2 protein (Smy2) interacts with mRNA-processing proteins through recognition of proline-rich sequences (PRS). Here, we describe the crystal structure of the GYF domain of Smy2 in association with a PRS from the yeast branch point binding protein (BBP/ScSF1). Complex formation requires that the beta-hairpin of the central PPGL motif of the ligand is accommodated by an extended hydrophobic cleft in the domain-a specificity feature that is maintained in the human protein GIGYF2. SILAC/MS experiments in combination with PRS site inhibition show that Smy2 associates with the Ccr4-NOT deadenylase complex, whereas GIGYF2 interacts not only with mRNA surveillance factors, but also with vesicular transport proteins and Atrophin-1. GIGYF2 is shown to associate with COPII-vesicle proteins and localize to the ER and Golgi in resting cells, whereas environmental challenge drives GIGYF2 into stress granules. The current study highlights the structural basis for PRS recognition by Smy2-type GYF domains, and implicates Smy2 and GIGYF2 in both mRNA processing and the secretory pathway.

To elucidate the function of the transcriptional coregulator PRMT1 (protein arginine methyltranferase 1) in interferon (IFN) signaling, we investigated the expression of STAT1 (signal transducer and activator of transcription) target genes in PRMT1-depleted cells. We show here that PRMT1 represses a subset of IFNginducible STAT1 target genes in a methyltransferase-dependent manner. These genes are also regulated by the STAT1 inhibitor PIAS1 (protein inhibitor of activated STAT1). PIAS1 is arginine methylated by PRMT1 in vitro as well as in vivo upon IFN treatment. Mutational and mass spectrometric analysis of PIAS1 identifies Arg 303 as the single methylation site. Using both methylation-deficient and methylation-mimicking mutants, we find that arginine methylation of PIAS1 is essential for the repressive function of PRMT1 in IFN-dependent transcription and for the recruitment of PIAS1 to STAT1 target gene promoters in the late phase of the IFN response. Methylation-dependent promoter recruitment of PIAS1 results in the release of STAT1 and coincides with the decline of STAT1-activated transcription. Accordingly, knockdown of PRMT1 or PIAS1 enhances the antiproliferative effect of IFNg. Our findings identify PRMT1 as a novel and crucial negative regulator of STAT1 activation that controls PIAS1-mediated repression by arginine methylation.[Keywords: Transcriptional repression; interferon signaling; STAT1; PIAS1; PRMT1; arginine methylation] Supplemental material is available at http://www.genesdev.org.

The two catalytic steps of splicing are a conceptually simple process that results in the RNA-mediated excision of introns (1). Complexity arises at the level of RNA-RNA and protein-RNA interactions that are needed for the correct spatial positioning of the critical bases, unwinding of RNA, and coupling of splicing to other processes as for example transcription (2, 3), nonsense-mediated mRNA decay (4), or mRNA transport (5, 6). Pre-mRNA splicing is catalyzed by the spliceosome, a dynamic macromolecular machine, which consists of the small nuclear ribonuclear particles (snRNPs), 1 U1, U2, U4/U6, and U5, and numerous non-snRNP proteins (7). The snRNPs interact with the intron in an ordered manner. First the U1 snRNP binds to the 5Ј splice site, whereas the U2 snRNP stably associates with the branch site forming complex A. Subsequently the tri-snRNP U4/U6.U5 is stably integrated to form complex B. This complex undergoes large structural rearrangements that lead to the formation of the activated spliceosomal complex B*. The catalytic steps of the transesterification then occur in complex C. Proline-rich sequences (PRS) are frequently found in spliceosomal proteins, and they are mostly assigned to unstructured regions of the corresponding full-length proteins (8). They serve as docking sites for so-called proline-rich sequence recognition domains (PRDs), and their interactions are characterized by low affinities and moderate specificities (9 -13). Several of the interactions between PRD and their proline-rich binding sites within the spliceosome have been characterized in vitro in great detail, but little is known about their functional importance. One interesting spliceosomal protein that mediates PRS interactions is CD2BP2/52K (14). It is a component of the U5 snRNP (15, 16) and contains a GYF adaptor domain that recognizes PRS via a set of conserved aromatic amino acids (17)(18)(19). A likely interaction partner of the GYF domain is the core splicing protein SmB/BЈ, which is present in all snRNPs. The GYF domain of CD2BP2/52K comprises a second interaction surface on a site opposite to the PRS binding epitope. This site is bound by the essential splicing protein U5-15K (16, 20). Considering that the U5 snRNP protein Prp6 interacts with the N-terminal part of CD2BP2/52K (16), several independent interactions seemingly contribute to the association of the protein with the U5 snRNP. However, for the GYF domain we could identify by phage display and peptide SPOT analysis additional interaction sites in other proteins suggesting that further "moonlighting" functions of the GYF domain might exist (21,22). This has set the basis for the current study where we performed pulldown experiments to define more generally the importance of CD2BP2/52K-GYF for the assembly of protein complexes. Utilizing the GYF domain fused to GST as bait, a large number of protein components of the U1, U2, U5, and the U4/U6.U5 tri-snRNP were co-precipitated. The association of most proteins was highly dependent on the PRS binding site in the GY...

The tumor maintenance protein Tsg101 has recently gained much attention because of its involvement in endosomal sorting, virus release, cytokinesis, and cancerogenesis. The ubiquitin-E2-like variant (UEV) domain of the protein interacts with proline-rich sequences of target proteins that contain P(S/T)AP amino acid motifs and weakly binds to the ubiquitin moiety of proteins committed to sorting or degradation. Here we performed peptide spot analysis and phage display to refine the peptide binding specificity of the Tsg101 UEV domain. A mass spectrometric proteomics approach that combines domain-based pulldown experiments, binding site inactivation, and stable isotope labeling by amino acids in cell culture (SILAC) was then used to delineate the relative importance of the peptide and ubiquitin binding sites.