Fragile X mental retardation protein (FMRP), the protein responsible for the fragile X syndrome, is an RNA-binding protein involved in localization and translation of neuronal mRNAs. One of the RNAs known to interact with FMRP is the dendritic non-translatable brain cytoplasmic RNA 1 BC1 RNA that works as an adaptor molecule linking FMRP and some of its regulated mRNAs. Here, we showed that the N terminus of FMRP binds strongly and specifically to BC1 and to its potential human analog BC200. This region does not contain a motif known to specifically recognize RNA and thus constitutes a new RNA-binding motif. We further demonstrated that FMRP recognition involves the 5 stem loop of BC1 and that this is the region that exhibits complementarity to FMRP target mRNAs, raising the possibility that FMRP plays a direct role in BC1/ mRNA annealing.The fragile X mental retardation protein (FMRP) 3 is the protein involved in the fragile X syndrome, the most common cause of inherited mental retardation. FMRP is highly expressed in neurons, where it is involved in mRNP transport and translation, two processes required for synaptic plasticity (1, 2). Thus, FMRP acts as a translational repressor both in vitro and in vivo (3)(4)(5)(6)(7)(8), and its effect is more pronounced at the synapses (7). The specific mechanism(s) through which FMRP regulates translation still remains to be understood; in particular, it is not clear whether the regulation occurs at the level of translation initiation (3), during the translation elongation phase (according to a "stalling polysomes" hypothesis) (8, 9), or both, depending on the different stages of development. With respect to mRNP transport, FMRP has both a nuclear localization signal (NLS) and a nuclear export signal (NES) and is capable of shuttling between the nucleus and the cytoplasm (10); it therefore seems likely that FMRP accompanies specific mRNAs from the nucleus to the cytoplasm. Furthermore, granules containing FMRP are transported to locations throughout the dendrite, where translation is regulated by synaptic activation (11), reminiscent of the granules in which mRNPs are thought to be transported. Indeed, mass spectrometric analysis of RNase-sensitive mRNP transport granules also identified, among several proteins involved in transport along the cytoskeleton, FMRP (12).As a protein involved in mRNP transport and regulation of translation, FMRP is expected to bind selectively to a subset of the mRNAs. Quite a variety of mRNAs have been identified in vitro and in vivo as potential targets of the entire FMRP (13-16), and it is still not clear how the mRNAs are recognized. There are at least three RNA elements that can direct FMRP binding (for a recent review, see Ref. 17). The first is a G-rich RNA structure called the G quartet (14,18,19), and the second consists of U-rich stretches (16). Thirdly, we have demonstrated that FMRP binds specifically to the non-coding RNA BC1, which in turn exhibits significant complementarity to and anneals with some mRNAs regulated by FMRP (7)...

Protein-RNA cross-linking combined with mass spectrometry is a powerful tool to elucidate hitherto noncharacterized protein-RNA contacts in ribonucleoprotein particles, as, for example, within spliceosomes. Here, we describe an improved methodology for the sequence analysis of purified peptide-RNA oligonucleotide cross-links that is based solely on MALDI-ToF mass spectrometry. The utility of this methodology is demonstrated on cross-links isolated from UV-irradiated spliceosomal particles; these were (1) [15.5K-61K-U4atac] small nuclear ribonucleoprotein (snRNP) particles prepared by reconstitution in vitro, and (2) U1 snRNP particles purified from HeLa cells. We show that the use of 2 0 ,4 0 ,6 0 -trihydroxyacetophenone (THAP) as MALDI matrix allows analysis of cross-linked peptide-RNA oligonucleotides in the reflectron mode at high resolution, enabling sufficient accuracy to assign unambiguously cross-linked RNA sequences. Most important, post-source decay (PSD) analysis under these conditions was successfully applied to obtain sequence information about the cross-linked peptide and RNA moieties within a single spectrum, including the identification of the actual cross-linking site. Thus, in U4atac snRNA we identified His270 in the spliceosomal U4/U6 snRNP-specific protein 61K (hPrp31p) cross-linked to U44; in the U1 snRNP we show that Leu175 of the U1 snRNP-specific 70K protein is cross-linked to U30 of U1 snRNA. This type of analysis is applicable to any type of RNP complex and may be expected to pave the way for the further analysis of protein-RNA complexes in much lower abundance and/or of cross-links that are obtained in low yield.

Protein-RNA interactions within ribonucleoprotein particles (RNPs) can be investigated by UV-induced crosslinking of proteins to their cognate RNAs and subsequent isolation and mass-spectrometric analysis of crosslinked peptide-RNA oligonucleotides. Because of the low crosslinking yield, a major challenge in protein-RNA UV crosslinking is the detection of the crosslinked species over the excess of non-crosslinked material, especially when complex systems (native RNPs) are investigated. Here, we applied a novel approach that uses on-line nanoLC-ESI-MS/MS to detect and subsequently sequence peptide-RNA oligonucleotide crosslinks from crude mixtures. To detect the crosslinks we made use of features shared by crosslinks and phosphopeptides, that is, the phosphate groups that both carry. A precursor ion scan for m/z 79 (negative-ion mode, Ϫve) is applied to selectively detect analytes bearing the phosphate-containing species (i.e., residual non-crosslinked RNA and peptide-RNA crosslinks) from crude mixtures and to determine their exact m/z values. On this basis, a multiple reaction monitoring (MRM) experiment monitors the expected decomposition from the different precursor charge states of the putative crosslinks to one of the four possible RNA nucleobases [m/z 112, 113, 136, 152 (positive-ion mode, ϩve)]. On detection, a high-quality MS/MS is triggered to establish the structure of the crosslink. In a feasibility study, we detected and subsequently sequenced peptide-RNA crosslinks obtained by UV-irradiation of (1) native U1 snRNPs and (2) Numerous MS-based proteomic approaches determined which proteins are associated with RNA [3][4][5][6][7]. However, these studies have yielded only limited information about which protein is in direct contact with the RNA. A straightforward method to determine this is crosslinking.Crosslinking between RNA and proteins can be achieved in different ways: (1) by incorporating base analogues (e.g., 5-bromo-2=-deoxyuridine [8], iododerivatives [9, 10], or 4-thio-uracil [11-13]) into the RNA, either site-specifically or randomly; (2) by chemical modification of the RNA backbone (acidophenacyl and benzophenone [14,15] [19]. The latter method uses the naturally occurring UV reactivity of the RNA nucleobases and has been successfully applied to various native protein-RNA complexes isolated from living cells [20 -22].Once the crosslink has been established, the main challenge is the identification of the crosslinked protein.

The kink-turn (k-turn), a new RNA structural motif found in the spliceosome and the ribosome, serves as a specific protein recognition element and as a structural building block. While the structure of the spliceosomal U4 snRNA k-turn/15.5K complex is known from a crystal structure, it is unclear whether the k-turn also exists in this folded conformation in the free U4 snRNA. Thus, we investigated the U4 snRNA k-turn by single-molecule FRET measurements in the absence and presence of the 15.5K protein and its dependence on the Na + and Mg 2+ ion concentration. We show that the unfolded U4 snRNA k-turn introduces a kink of 85°-15°in an RNA double helix. While Na + and Mg 2+ ions induce this more open conformation of the k-turn, binding of the 15.5K protein was found to induce the tightly kinked conformation in the RNA that increases the kink to 52°-15°. By comparison of the measured FRET distances with a computer-modeled structure, we show that this strong kink is due to the kturn motif adopting its folded conformation. Thus, in the free U4 snRNA, the k-turn exists only in an unfolded conformation, and its folding is induced by binding of the 15.5K protein.

Direct UV cross-linking combined with mass spectrometry (MS) is a powerful tool to identify hitherto non-characterized protein–RNA contact sites in native ribonucleoprotein particles (RNPs) such as the spliceosome. Identification of contact sites after cross-linking is restricted by: (i) the relatively low cross-linking yield and (ii) the amount of starting material available for cross-linking studies. Therefore, the most critical step in such analyses is the extensive purification of the cross-linked peptide–RNA heteroconjugates from the excess of non-crosslinked material before MS analysis. Here, we describe a strategy that combines small-scale reversed-phase liquid chromatography (RP-HPLC) of UV-irradiated and hydrolyzed RNPs, immobilized metal-ion affinity chromatography (IMAC) to enrich cross-linked species and their analysis by matrix-assisted laser desorption/ionisation (MALDI) MS(/MS). In cases where no MS/MS analysis can be performed, treatment of the enriched fractions with alkaline phosphatase leads to unambiguous identification of the cross-linked species.We demonstrate the feasibility of this strategy by MS analysis of enriched peptide–RNA cross-links from UV-irradiated reconstituted [15.5K-61K-U4atac snRNA] snRNPs and native U1 snRNPs. Applying our approach to a partial complex of U2 snRNP allowed us to identify the contact site between the U2 snRNP-specific protein p14/SF3b14a and the branch-site interacting region (BSiR) of U2 snRNA.

Mass spectrometry allows the elucidation of molecular details of the interaction domains of the individual components in macromolecular complexes subsequent to cross-linking of the individual components. Here, we applied chemical and UV cross-linking combined with tandem mass-spectrometric analysis to identify contact sites of the nuclear import adaptor snurportin 1 to the small ribonucleoprotein particle U1 snRNP in addition to the known interaction of m3G cap and snurportin 1. We were able to define previously unknown sites of protein–protein and protein–RNA interactions on the molecular level within U1 snRNP. We show that snurportin 1 interacts with its central m3G-cap-binding domain with Sm proteins and with its extreme C-terminus with stem-loop III of U1 snRNA. The crosslinking data support the idea of a larger interaction area between snurportin 1 and U snRNPs and the contact sites identified prove useful for modeling the spatial arrangement of snurportin 1 domains when bound to U1 snRNP. Moreover, this suggests a functional nuclear import complex that assembles around the m3G cap and the Sm proteins only when the Sm proteins are bound and arranged in the proper orientation to the cognate Sm site in U snRNA.

Mass spectrometry is a powerful tool for the analysis of biomolecules, proteins, nucleic acids, carbohydrates, lipids. In combination with genome sequences that are available in the databases, it has proven to be the most straightforward and sensitive technique for the sequence analysis and hence the identification of protein components in the cells, their (post)translational modifications, and their relative and absolute abundance. In addition, mass spectrometric methods are successfully applied for the structural analysis of biomolecules (i.e., deciphering molecule-ligand interactions and spatial quartenary arrangements of molecule complexes). We describe a methodology for the mass spectrometric analysis of protein-RNA contact sites in purified ribonucleoprotein (RNP) particles. The method comprises ultraviolet (UV) crosslinking of proteins to RNA, hydrolysis of the protein and RNA moieties, isolation of cross-linked peptide-RNA oligonucleotides, MALDI (matrix-assisted laser desorption/ionization) mass spectrometry of the isolated conjugates to determine the sequence of the crosslinked peptide and RNA part. The utility of this methodology is demonstrated on crosslinks isolated from UV-irradiated spliceosomal particles; these were [15.5 K-61 K-U4atac] small nuclear ribonucleoprotein (snRNP) particles prepared by reconstitution in vitro and U1 snRNP particles purified from HeLa cells.

Proteomics is rapidly developing into a routine approach for protein analysis in many laboratories. The series of European-wide Summer Schools 'Proteomics Basics' (http://www.proteomic-basics.eu/) aims at teaching of comprehensive knowledge in proteomics research and applied technologies for master and graduate students and postdocs currently moving into the field of proteomic research. In the next 3 years the series will cover the theoretical basis of the fundamental topics in the various areas of proteomic analysis, i.e. sample preparation and handling, mass spectrometry, post-translational modifications and quantitation given by leading experts in the field. This summer school series embodies a unique advantage in comparison with conventional scientific meetings and university curricula: internationally renowned experts will give a detailed perspective view of the fundamentals of their particular proteome research area, something which is usually not encountered at conferences and congresses. Here, we give a report on the first European Summer School 'Sample Preparation and Handling' within the series 'Proteomic Basics' that was held at the monastery in Neustift close to Bressanone/Brixen, Italy from August 12 to 18, 2007.