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.

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 ∼3 MDa complex in the cytoplasm and a 20-fold smaller complex of ∼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.

Diffusion and segmental dynamics of the double-stranded -phage DNA polymer are quantitatively studied over the transition range from stiff to semiflexible chains. Spectroscopy of fluorescence fluctuations of single-end fluorescently labeled monodisperse DNA fragments unambiguously shows that doublestranded DNA in the length range of 10 2 -2 10 4 base pairs behaves as a semiflexible polymer with segmental dynamics controlled by hydrodynamic interactions. DOI: 10.1103/PhysRevLett.97.258101 PACS numbers: 87.14.Gg, 82.35.Lr, 87.15.He, 87.15.Vv The dynamic behavior of individual macromolecules in solution is governed by chain connectivity and hydrodynamic interactions [1]. Understanding polymer dynamics and quantitative verification of polymer theories requires detailed information on segmental motion of individual polymer molecules. However, the classical experimental techniques, such as dynamic light scattering (DLS) or transient electric birefringence (TEB), predominantly deliver information on large-scale shape fluctuations of macromolecules, and development of new experimental approaches providing detailed information on internal dynamics of individual molecules is of high importance.Precise experiments in polymer physics are impossible without well-defined monodisperse polymer samples covering a wide range of molecular weights. The recent progress in molecular biotechnology resulted in a variety of techniques to produce monodisperse DNA fragments, which stimulated the use of DNA as a model compound in studies of polymer dynamics in solution [2]. Doublestranded DNA (dsDNA) is a biopolymer characterized by a large persistence length l p 50 nm [3]. As a result, dsDNA fragments exhibit rodlike, semiflexible, or even flexible polymer behavior, depending on their length. Thus, simple generic models of polymer dynamics [1] are not expected to provide a quantitative description of dsDNA behavior, and more advanced models accounting for the persistence of the polymer chain [4] are required.The dynamics of dsDNA in solution has been a subject of a number of experimental investigations carried out by various techniques, including DLS [5], single-molecule fluorescence microscopy [6], electrophoresis [7], and TEB [8]. Fluorescence correlation spectroscopy (FCS) [9], a single-molecule technique that can provide more detailed information on the macromolecular dynamics than the classical ensemble-based methods, has been recently applied to investigation of DNA in solution [10 -12], which has lead to a controversy whether dsDNA dynamics in dilute solution is controlled by hydrodynamic interactions [10,12] or not [11]. Apart from this controversy, for different reasons, none of these FCS-based experiments produced parameters of DNA dynamics. Generally, to the best of our knowledge, no experimental studies were published so far where diffusion and intramolecular dynamics of dsDNA have been simultaneously investigated over the transition range from stiff to semiflexible chains. In the present Letter, we fill this gap and car...

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.

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.