Molecular dissection of step 2 catalysis of yeast pre-mRNA splicing investigated in a purified system.

Step 2 catalysis of pre-mRNA splicing entails the excision of the intron and ligation of the 5' and 3' exons. The tasks of the splicing factors Prp16, Slu7, Prp18, and Prp22 in the formation of the step 2 active site of the spliceosome and in exon ligation, and the timing of their recruitment, remain poorly understood. Using a purified yeast in vitro splicing system, we show that only the DEAH-box ATPase Prp16 is required for formation of a functional step 2 active site and for exon ligation. Efficient docking of the 3' splice site (3'SS) to the active site requires only Slu7/Prp18 but not Prp22. Spliceosome remodeling by Prp16 appears to be subtle as only the step 1 factor Cwc25 is dissociated prior to step 2 catalysis, with its release dependent on docking of the 3'SS to the active site and Prp16 action. We show by fluorescence cross-correlation spectroscopy that Slu7/Prp18 and Prp16 bind early to distinct, low-affinity binding sites on the step-1-activated B* spliceosome, which are subsequently converted into high-affinity sites. Our results shed new light on the factor requirements for step 2 catalysis and the dynamics of step 1 and 2 factors during the catalytic steps of splicing.

Prp2-mediated protein rearrangements at the catalytic core of the spliceosome as revealed by dcFCCS.

The compositional and conformational changes during catalytic activation of the spliceosome promoted by the DEAH box ATPase Prp2 are only poorly understood. Here, we show by dual-color fluorescence cross-correlation spectroscopy (dcFCCS) that the binding affinity of several proteins is significantly changed during the Prp2-mediated transition of precatalytic B(act) spliceosomes to catalytically activated B* spliceosomes from Saccharomyces cerevisiae. During this step, several proteins, including the zinc-finger protein Cwc24, are quantitatively displaced from the B* complex. Consistent with this, we show that Cwc24 is required for step 1 but not for catalysis per se. The U2-associated SF3a and SF3b proteins Prp11 and Cus1 remain bound to the B* spliceosome under near-physiological conditions, but their binding is reduced at high salt. Conversely, high-affinity binding sites are created for Yju2 and Cwc25 during catalytic activation, consistent with their requirement for step 1 catalysis. Our results suggest high cooperativity of multiple Prp2-mediated structural rearrangements at the spliceosome's catalytic core. Moreover, dcFCCS represents a powerful tool ideally suited to study quantitatively spliceosomal protein dynamics in equilibrium.

Intracellular localization and routing of miRNA and RNAi pathway components.

Several different pathways, generally termed RNA silencing pathways, utilize small RNA molecules guiding sequence-specific silencing effects of ribonucleoprotein effector complexes, traditionally termed RNA-induced silencing complex (RISC). Three RNA silencing pathways were recognized in mammalian cells: RNA interference (RNAi), where short RNAs produced from long double-stranded RNA guide cleavage of cognate mRNAs, microRNA (miRNA) pathway, where endogenously-encoded miRNAs typically induce translational repression, and piRNA pathway, where piRNAs (PIWI-associated RNAs) guide repression of repetitive sequences in the germline. Originally, RNAi and miRNA pathways were thought to act in the cytoplasm, however, there is a growing body of evidence that these pathways also have a nuclear component. This text reviews the current evidence concerning nuclear localization and function of miRNA and RNAi pathway components. We provide evidence that TRBP, Dicer and AGO2, proteins found in the RISC-loading complex (RLC) and RISC itself, are present in the nucleus. Nonetheless, fully functional RLC is not found in the nuclear compartment which is consistent with the recent findings obtained by Fluorescence Cross-Correlation Spectroscopy experiments illustrating that RISC is specifically loaded within the cytoplasm and shuttles subsequently between the nuclear and cytoplasmic compartment, thereby allowing small RNA gene regulation in both compartments. The function of nuclear TRBP and Dicer proteins remains elusive. We also discuss the consequences of nucleotide analogs introduced into siRNAs which can severely interfere with the natural cytoplasmic localization mediated by Exportin-5 which is required for efficient RISC loading in the cytoplasm.

Fluorescence cross-correlation spectroscopy reveals mechanistic insights into the effect of 2'-O-methyl modified siRNAs in living cells.

RNA interference (RNAi) offers a powerful tool to specifically direct the degradation of complementary RNAs, and thus has great therapeutic potential for targeting diseases. Despite the reported preferences of RNAi, there is still a need for new techniques that will allow for a detailed mechanistic characterization of RNA-induced silencing complex (RISC) assembly and activity to further improve the biocompatibility of modified siRNAs. In contrast to previous reports, we investigated the effects of 2'-O-methyl (2'OMe) modifications introduced at specific positions within the siRNA at the early and late stages of RISC assembly, as well as their influence on target recognition and cleavage directly in living cells. We found that six to 10 2'OMe nucleotides on the 3'-end inhibit passenger-strand release as well as target-RNA cleavage without changing the affinity, strand asymmetry, or target recognition. 2'OMe modifications introduced at the 5'-end reduced activated RISC stability, whereas incorporations at the cleavage site showed only minor effects on passenger-strand release when present on the passenger strand. Our new fluorescence cross-correlation spectroscopy assays resolve different steps and stages of RISC assembly and target recognition with heretofore unresolved detail in living cells, which is needed to develop therapeutic siRNAs with optimized in vivo properties.

Importin 8 is a gene silencing factor that targets argonaute proteins to distinct mRNAs.

Small regulatory RNAs including small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide Argonaute (Ago) proteins to specific target RNAs leading to mRNA destabilization or translational repression. Here, we report the identification of Importin 8 (Imp8) as a component of miRNA-guided regulatory pathways. We show that Imp8 interacts with Ago proteins and localizes to cytoplasmic processing bodies (P bodies), structures involved in RNA metabolism. Furthermore, we detect Ago2 in the nucleus of HeLa cells, and knockdown of Imp8 reduces the nuclear Ago2 pool. Using immunoprecipitations of Ago2-associated mRNAs followed by microarray analysis, we further demonstrate that Imp8 is required for the recruitment of Ago protein complexes to a large set of Ago2-associated target mRNAs, allowing for efficient and specific gene silencing. Therefore, we provide evidence that Imp8 is required for cytoplasmic miRNA-guided gene silencing and affects nuclear localization of Ago proteins.

Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy reveal the cytoplasmic origination of loaded nuclear RISC in vivo in human cells.

Studies of RNA interference (RNAi) provide evidence that in addition to the well-characterized cytoplasmic mechanisms, nuclear mechanisms also exist. The mechanism by which the nuclear RNA-induced silencing complex (RISC) is formed in mammalian cells, as well as the relationship between the RNA silencing pathways in nuclear and cytoplasmic compartments is still unknown. Here we show by applying fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS) in vivo that two distinct RISC exist: a large approximately 3 MDa complex in the cytoplasm and a 20-fold smaller complex of approximately 158 kDa in the nucleus. We further show that nuclear RISC, consisting only of Ago2 and a short RNA, is loaded in the cytoplasm and imported into the nucleus. The loaded RISC accumulates in the nucleus depending on the presence of a target, based on an miRNA-like interaction with impaired cleavage of the cognate RNA. Together, these results suggest a new RISC shuttling mechanism between nucleus and cytoplasm ensuring concomitant gene regulation by small RNAs in both compartments.

Characterization of protein dynamics in asymmetric cell division by scanning fluorescence correlation spectroscopy.

The development and differentiation of complex organisms from the single fertilized egg is regulated by a variety of processes that all rely on the distribution and interaction of proteins. Despite the tight regulation of these processes with respect to temporal and spatial protein localization, exact quantification of the underlying parameters, such as concentrations and distribution coefficients, has so far been problematic. Recent experiments suggest that fluorescence correlation spectroscopy on a single molecule level in living cells has great promise in revealing these parameters with high precision. The optically challenging situation in multicellular systems such as embryos can be ameliorated by two-photon excitation, where scattering background and cumulative photobleaching is limited. A more severe problem is posed by the large range of molecular mobilities observed at the same time, as standard FCS relies strongly on the presence of mobility-induced fluctuations. In this study, we overcame the limitations of standard FCS. We analyzed in vivo polarity protein PAR-2 from eggs of Caenorhabditis elegans by beam-scanning FCS in the cytosol and on the cortex of C. elegans before asymmetric cell division. The surprising result is that the distribution of PAR-2 is largely uncoupled from the movement of cytoskeletal components of the cortex. These results call for a more systematic future investigation of the different cortical elements, and show that the FCS technique can contribute to answering these questions, by providing a complementary approach that can reveal insights not obtainable by other techniques.

Cellular dynamics of Ku: characterization and purification of Ku-eGFP.

Ku is a predominantly nuclear protein that functions as a DNA double-strand-break (DSB) binding protein and regulatory subunit of the DNA-dependent protein kinase (DNA-PK). DNA-PK is involved in synapsis and remodeling of broken DNA ends during nonhomologous end-joining (NHEJ) of DNA DSBs. It has also recently been demonstrated that Ku plays roles in cytoplasmic and membrane processes, namely: interaction with matrix metalloproteinase 9, acting as a co-receptor for parvoviral infection, and also interacting with cell polarity protein, Par3. We present a method for creating stable expression of Ku-eGFP in CHO cells and extend the procedure to purify Ku-eGFP for in vitro assaying. We demonstrated that Ku-eGFP localizes to the nucleus of HeLa cells upon microinjection into the cytoplasm as well as localizing to laser induced DNA damage. We also characterized the diffusional dynamics of Ku in the nucleus and in the cytoplasm using fluorescence correlation spectroscopy (FCS). The FCS data suggest that whereas the majority of Ku (70%) in the nucleus is mobile and freely diffusing, in a cellular context, there also exists a significant slow process fraction (30%). Strikingly, in the cytoplasm, this immobile/slow moving fraction is even more pronounced (45%).

In situ fluorescence analysis demonstrates active siRNA exclusion from the nucleus by Exportin 5.

Two types of short double-stranded RNA molecules, namely microRNAs (miRNAs) and short interfering RNAs (siRNAs), have emerged recently as important regulators of gene expression. Although these molecules show similar sizes and structural features, the mechanisms of action underlying their respective target silencing activities appear to differ: siRNAs act primarily through mRNA degradation, whereas most miRNAs appear to act primarily through translational inhibition. Our understanding of how these overlapping pathways are differentially regulated within the cell remains incomplete. In the present work, quantitative fluorescence microscopy was used to study how siRNAs are processed within human cells. We found that siRNAs are excluded from non-nucleolar areas of the nucleus in an Exportin-5 dependent process that specifically recognizes key structural features shared by these and other small RNAs such as miRNAs. We further established that the Exportin-5-based exclusion of siRNAs from the nucleus can, when Exp5 itself is inhibited, become a rate-limiting step for siRNA-induced silencing activity. Exportin 5 therefore represents a key point of intersection between the siRNA and miRNA pathways, and, as such, is of fundamental importance for the design and interpretation of RNA interference experimentation.

c-Cbl binds to tyrosine-phosphorylated neurotrophin receptor p75 and induces its ubiquitination.

The p75 neurotrophin receptor (p75NTR) has dual functions in cell survival and cell death but its intracellular signalling pathways are not understood. Here we describe that in rat brain and in pervanadate-stimulated PCNA and HEK293 cells p75NTR is phosphorylated at a single tyrosine residue within the cytosolic C-terminus. Phosphorylated tyrosine 308 constitutes a binding site for the ubiquitin ligase c-Cbl. This interaction is a prerequisite for ubiquitination of p75NTR. Our data suggest a c-Cbl-dependent ubiquitination of p75NTR involved in the regulation of p75NTR signalling.