Identification of tobacco proteins associated with the stem-loop 1 RNAs of <Emphasis Type="Italic">Potato virus X</Emphasis>

Potato virus X (PVX) contains five viral proteins as well as cis-acting elements like stem-loop 1 (SL1) RNAs at the 5′ region. SL1 RNAs are involved in PVX RNA replication, encapsidation, translation, and cell-to-cell movement. In this study, we performed two-dimensional electrophoresis Northwestern blot analysis and matrix-assisted laser desorption ionization time of flight mass spectrometry and identified 24 tobacco proteins that interact with SL1 RNAs. Interestingly, one-third of the identified host proteins have been shown to interact with many plant viral proteins. In addition, we demonstrated that PVX capsid protein can bind to both SL1(+/−) RNAs. We further selected three Nicotiana benthamiana proteins including NbMPB2Cb, NbMBF1, and NbCPIP2a, to confirm results of Northwestern blot analysis. Electrophoretic mobility shift assay showed that NbMPB2Cb and NbMBF1 bind to both SL1(+/−) RNAs in vitro. In contrast, NbCPIP2a interacts only SL1(+) RNA. Taken together, we provide a list of host proteins interacting with PVX SL1 RNAs, which would be good candidates for the study of viral RNA-host protein interaction.     

Spliced leader RNA of trypanosomes: in vivo mutational analysis reveals extensive and distinct requirements for trans splicing and cap4 formation.

In trypanosomes mRNAs are generated through trans splicing. The spliced leader (SL) RNA, which donates the 5'-terminal mini-exon to each of the protein coding exons, plays a central role in the trans splicing process. We have established in vivo assays to study in detail trans splicing, cap4 modification, and RNP assembly of the SL RNA in the trypanosomatid species Leptomonas seymouri. First, we found that extensive sequences within the mini-exon are required for SL RNA function in vivo, although a conserved length of 39 nt is not essential. In contrast, the intron sequence appears to be surprisingly tolerant to mutation; only the stem-loop II structure is indispensable. The asymmetry of the sequence requirements in the stem I region suggests that this domain may exist in different functional conformations. Second, distinct mini-exon sequences outside the modification site are important for efficient cap4 formation. Third, all SL RNA mutations tested allowed core RNP assembly, suggesting flexible requirements for core protein binding. In sum, the results of our mutational analysis provide evidence for a discrete domain structure of the SL RNA and help to explain the strong phylogenetic conservation of the mini-exon sequence and of the overall SL RNA secondary structure; they also suggest that there may be certain differences between trans splicing in nematodes and trypanosomes. This approach provides a basis for studying RNA-RNA interactions in the trans spliceosome.     

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.¹ The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 μm wavelength utilize the sudden excitation of water that results in phase explosion.² Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.^3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.¹² This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.     

In vivo structural analysis of spliced leader RNAs in Trypanosoma brucei and Leptomonas collosoma: a flexible structure that is independent of cap4 methylations.

The formation of the mRNA 5' end in trypanosomatid protozoa is carried out by trans-splicing, which transfers a spliced leader (SL) sequence and its hypermethylated cap (cap4) from the SL RNA to the pre-mRNA. Previous in vitro studies with synthetic uncapped RNAs have shown that the SL sequence of Leptomonas collosoma can assume two alternate conformations, Form 1 and Form 2, with Form 1 being the dominant one. To gain information about the structure of the SL RNA in vivo, in its protein-rich environment, we have used permeable Trypanosoma brucei and L. collosoma cells for chemical modification experiments. We introduce the use in vivo of the water-soluble reagents CMCT and kethoxal. In contrast to the in vitro results, the Form 2 secondary structure predominates. However, there are chemically accessible regions that suggest conformational flexibility in SL RNPs and a chemically inaccessible region suggestive of protection by protein or involvement in tertiary interactions. Using complementary 2'-O-methyl RNA oligonucleotides, we show that T. brucei SL RNA can be induced to switch conformation in vivo. SL RNA stripped of proteins and probed in vitro does not display the same Form 2 bias, indicating that SL RNA structure is determined, at least in part, by its RNP context. Finally, the methyl groups of the cap4 do not seem to affect the secondary structure of T. brucei SL RNA, as shown by chemical modification of undermethylated SL RNA probed in vivo.     

On the role of exon and intron sequences in trans-splicing utilization and cap 4 modification of the trypanosomatid Leptomonas collosoma SL RNA

In trypanosomatid protozoa the biogenesis of mature mRNA involves addition of the spliced leader (SL) sequence from the SL RNA to polycistronic pre-mRNA via trans-splicing. Here we present a mutational analysis of the trypanosomatid Leptomonas collosoma SL RNA to further our understanding of its functional domains important for trans-splicing utilization. Mutant SL RNAs were analyzed for defects in modification of the hypermethylated cap structure (cap 4) characteristic of trypanosomatid SL RNAs, for defects in the first step of the reaction and overall utilization in trans-splicing. Single substitution of the cap 4 nucleotides led to undermethylation of the cap 4 structure, and these mutants were all impaired in their utilization in trans-splicing. Abrogation of the sequence of the Sm-like site and sequences downstream to it also showed cap modification and trans-splicing defects, thus providing further support for a functional linkage between cap modifications and trans-splicing. Further, we report that in L. collosoma both the exon and intron of the SL RNA contribute information for efficient function of the SL RNA in trans-splicing. This study, however, did not provide support for the putative SL RNA-U6 small nuclear RNA (snRNA) interaction at the Sm site like in the nematodes, suggesting differences in the bridging role of U6 in the two trans-splicing systems.     

Structural determination and chemical esterification of the sophorolipids produced by <Emphasis Type="Italic">Candida bombicola</Emphasis> grown on glucose and α-linolenic acid

The extracellular surface-active glycolipids produced by the yeast, Candida bombicola when grown on glucose and α-linolenic acid, were analyzed by HPLC with electro-spray ionization (ESI−MS) and collision-induced dissociation mass spectrometry. The analysis confirmed that the sophorolipid (SL) mixture contained three different forms of C18:3 SL molecules: free acid, lactone and a diacetylated lactone, which has not been reported previously. Also a minor amount of diacetylated lactone form of C18:1 SL was detected. Further, the SL mixture was subjected to chemical esterification reaction with sodium methoxide. The reaction product was analyzed with ESI−MS and confirmed to be the single homogenous esterified product containing C18:3 moieties in its fatty acid chain.     

Changes in 7SL RNA conformation during the signal recognition particle cycle.

The structure of 7SL RNA has been probed by chemical modification followed by primer extension, using four substrates: (i) naked 7SL RNA; (ii) free signal recognition particle (SRP); (iii) polysome bound SRP; and (iv) membrane bound SRP. Decreasing sensitivity to chemical modification between these different substrates suggests regions on 7SL RNA that: bind proteins associated with SRP might interact with ribosomes; and are protected by binding to membranes. Other areas increase in chemical sensitivity, exemplified by a tertiary interaction present in naked 7SL RNA but not in free SRP. Such changes suggest that 7SL RNA changes its conformation during the SRP cycle. These conformational changes could be a necessary component to move through the SRP cycle from one stage to the next.     

Characterization of EGF coupling to aminated silicone rubber surfaces

Tethering of growth factors to biomaterial substrates via a polyethylene glycol (PEG) spacer has been established as a means of controlling dosage and conformation of the protein at the material surface, while retaining biological activity. However, the extent of modification through a comparison of bound versus unbound protein has not generally been characterized. In this work, covalent tethering of epidermal growth factor (EGF) to allylamine plasma modified polydimethylsiloxane (PDMS) substrates is characterized to determine the nature of the bound growth factor and to optimize the conditions for the reaction. Tethering is achieved via conjugation of EGF with homobifunctional N-hydroxysuccinimide (NHS) ester of PEG-butanoic acid (SBA2-PEG) in solution, followed by exposure of the pegylated EGF to the aminated surfaces (solution first reaction). SDS-PAGE analysis indicates that a low ratio of EGF:PEG is required to maximize the yield of the EGF-PEG reaction; a relatively short reaction time is needed to limit hydrolysis of the NHS ester. With increasing amounts of PEG and a higher reaction time, a higher fraction of the EGF can be covalently tethered to the surfaces, as shown by binding of 125I-labeled EGF and subsequent washing with sodium dodecyl sulfate (SDS) to remove adsorbed protein. However, even under the optimal reaction conditions established by the SDS-PAGE analysis, higher molecular weight EGF-PEG complexes are observed by SDS-PAGE and matrix-assisted laser desorption/ionization (MALDI). The presence of these complexes, as well as unreacted growth factor, can lead to a surface of heterogeneous composition. While these surfaces were found to have biological activity, stimulating the adhesion and growth of corneal epithelial cells versus PDMS controls, further optimization of reaction conditions, including the use of a homobifunctional PEG linker and possibly separation of reaction species are required to achieve a uniformly active and well-defined biomaterial surface.     

Silencing of Sm proteins in Trypanosoma brucei by RNA interference captured a novel cytoplasmic intermediate in spliced leader RNA biogenesis

In Trypanosoma brucei the small nuclear (sn) RNAs U1, U2, U4, and U5, as well as the spliced leader (SL) RNA, bind the seven Sm canonical proteins carrying the consensus Sm motif. To determine the function of these proteins in snRNA and SL RNA biogenesis, two of the Sm core proteins, SmE and SmD1, were silenced by RNAi. Surprisingly, whereas the level of all snRNAs, including U1, U2, U4, and U5 was reduced during silencing, the level of SL RNA was dramatically elevated, but the levels of U6 and spliced leader-associated RNA (SLA1) remained unchanged. The SL RNA that had accumulated in silenced cells lacked modification at the cap4 nucleotide but harbored modifications at the cap1 and cap2 nucleotides and carried the characteristic psi. This SL RNA possessed a longer tail and had accumulated in the cytoplasm in 10 and 50 S particles that were found by in situ hybridization to be present in "speckles." We propose a model for SL RNA biogenesis involving a cytoplasmic phase and suggest that the trypanosome-specific "cap4" nucleotides function as a signal for export and import of SL RNA out and into the nucleus. The SL RNA biogenesis pathway differs from that of U sn ribonucleoproteins (RNPs) in that it is the only RNA that binds Sm proteins that were stabilized under Sm depletion in a novel RNP, which we termed SL RNP-C.     

Imaging lipids in biological samples with surface-assisted laser desorption/ionization mass spectrometry: A concise review of the last decade

Knowing the spatial location of the lipid species present in biological samples is of paramount importance for the elucidation of pathological and physiological processes. In this context, mass spectrometry imaging (MSI) has emerged as a powerful technology allowing the visualization of the spatial distributions of biomolecules, including lipids, in complex biological samples. Among the different ionization methods available, the emerging surface-assisted laser desorption/ionization (SALDI) MSI offers unique capabilities for the study of lipids. This review describes the specific advantages of SALDI-MSI for lipid analysis, including the ability to perform analyses in both ionization modes with the same nanosubstrate, the detection of lipids characterized by low ionization efficiency in MALDI-MS, and the possibilities of surface modification to improve the detection of lipids. The complementarity of SALDI and MALDI-MSI is also discussed. Finally, this review presents data processing strategies applied in SALDI-MSI of lipids, as well as examples of applications of SALDI-MSI in biomedical lipidomics.     

Identifying requirements for RSK2 specific inhibitors

Identifying isoform-specific inhibitors for closely related kinase family members remains a substantial challenge. The necessity for achieving this specificity is exemplified by the RSK family, downstream effectors of ERK1/2, which have divergent physiological effects. The natural product, SL0101, a flavonoid glycoside, binds specifically to RSK1/2 through a binding pocket generated by an extensive conformational rearrangement within the RSK N-terminal kinase domain (NTKD). In modelling experiments a single amino acid that is divergent in RSK3/4 most likely prevents the required conformational rearrangement necessary for SL0101 binding. Kinetic analysis of RSK2 association with SL0101 and its derivatives identified that regions outside of the NTKD contribute to stable inhibitor binding. An analogue with an n-propyl-carbamate at the 4” position on the rhamnose moiety was identified that forms a highly stable inhibitor complex with RSK2 but not with RSK1. These results identify a SL0101 modification that will aid the identification of RSK2 specific inhibitors.     

The 2'-O-ribose methyltransferase for cap 1 of spliced leader RNA and U1 small nuclear RNA in Trypanosoma brucei

mRNA cap 1 2'-O-ribose methylation is a widespread modification that is implicated in processing, trafficking, and translational control in eukaryotic systems. The eukaryotic enzyme has yet to be identified. In kinetoplastid flagellates trans-splicing of spliced leader (SL) to polycistronic precursors conveys a hypermethylated cap 4, including a cap 0 m7G and seven additional methylations on the first 4 nucleotides, to all nuclear mRNAs. We report the first eukaryotic cap 1 2'-O-ribose methyltransferase, TbMTr1, a member of a conserved family of viral and eukaryotic enzymes. Recombinant TbMTr1 methylates the ribose of the first nucleotide of an m7G-capped substrate. Knockdowns and null mutants of TbMTr1 in Trypanosoma brucei grow normally, with loss of 2'-O-ribose methylation at cap 1 on substrate SL RNA and U1 small nuclear RNA. TbMTr1-null cells have an accumulation of cap 0 substrate without further methylation, while spliced mRNA is modified efficiently at position 4 in the absence of 2'-O-ribose methylation at position 1; downstream cap 4 methylations are independent of cap 1. Based on TbMTr1-green fluorescent protein localization, 2'-O-ribose methylation at position 1 occurs in the nucleus. Accumulation of 3'-extended SL RNA substrate indicates a delay in processing and suggests a synergistic role for cap 1 in maturation.     

Sophorolipids: improvement of the selective production by Starmerella bombicola through the design of nutritional requirements

Microtiter plates were used as minireactors to study Starmerella bombicola growth and sophorolipid (SL) production. Compositional analysis of SL mixtures by liquid chromatography with electrospray ionization tandem mass spectrometry showed similar results on SLs produced using the laboratory scale (shake flask) and the microscale (24-well microtiter plates (MTP)) approach. MTP suitability on SL production was proven, being this approach, especially advantageous on SL screening. Several hydrophilic carbon sources, hydrophobic co-substrates and nitrogen sources were supplied to culture media, and their influence on SL production was evaluated. The selection of specific hydrophobic co-substrate and nitrogen sources influenced the ratio acidic/lactonic SLs. In fact, it was observed that the production of acidic C18:1 diacetylated hydroxy fatty acid SLs was favoured when culture media was supplied with avocado, argan, sweet almond and jojoba oil or when NaNO₃ was supplied instead of urea. This last case was observed after 144 h of cultivation. A new SL, lactonic C18:3 hydroxy fatty acid diacetylated SL, was detected when borage and onagra oils were used individually as co-substrates. Overall results indicated the potential of the selective production of different and new sophorolipids by Starmerella bombicola based on the selection of carbon and nitrogen sources to culture media.     

Generic bioaffinity silicone surfaces

Synthetic polymer surfaces require surface modification to improve biocompatibility. A generic route to biocompatible silicone elastomers is described involving high yield surface functionalization of standard silicones with hydrosilanes, hydrosilylation using asymmetric, allyl-, NSC-terminated PEO of narrow molecular weight, and covalent modification in one step with amine-containing biological molecules including oligopeptides (YIGSR, RGDS), proteins (EGF, albumin, fibrinogen, mucin), and glycosaminoglycans (heparin). Efficient, high-density binding (e.g., 0.2 EGF molecules/nm2) was demonstrated using radiolabeling studies. The resulting surfaces were demonstrated to be biocompatible by further reaction with biomolecules, for example, thrombosis suppression on surfaces modified by heparin + ATIII, and the formation of confluent corneal epithelial cell layers on EGF, RGDS, or YIGSR surfaces.     

Protein mass analysis of histones

Posttranslational modification of chromatin-associated proteins, including histones and high-mobility-group (HMG) proteins, provides an important mechanism to control gene expression, genome integrity, and epigenetic inheritance. Protein mass analysis provides a rapid and unbiased approach to monitor multiple chemical modifications on individual molecules. This review describes methods for acid extraction of histones and HMG proteins, followed by separation by reverse-phase chromatography coupled to electrospray ionization mass spectrometry (LC/ESI-MS). Posttranslational modifications are detected by analysis of full-length protein masses. Confirmation of protein identity and modification state is obtained through enzymatic digestion and peptide sequencing by MS/MS. For differentially modified forms of each protein, the measured intensities are semiquantitative and allow determination of relative abundance and stoichiometry. The method simultaneously detects covalent modifications on multiple proteins and provides a facile assay for comparing chromatin modification states between different cell types and/or cellular responses.