Direct Access Route Research Articles

Transcriptome-wide analysis of alternative routes for RNA substrates into the exosome complex.

The RNA exosome complex functions in both the accurate processing and rapid degradation of many classes of RNA. Functional and structural analyses indicate that RNA can either be threaded through the central channel of the exosome or more directly access the active sites of the ribonucleases Rrp44 and Rrp6, but it was unclear how many substrates follow each pathway in vivo. We used CRAC (UV crosslinking and analysis of cDNA) in growing cells to identify transcriptome-wide interactions of RNAs with the major nuclear exosome-cofactor Mtr4 and with individual exosome subunits (Rrp6, Csl4, Rrp41 and Rrp44) along the threaded RNA path. We compared exosome complexes lacking Rrp44 exonuclease activity, carrying a mutation in the Rrp44 S1 RNA-binding domain predicted to disfavor direct access, or with multiple mutations in Rrp41 reported to impede RNA access to the central channel in vitro. Preferential use of channel-threading was seen for mRNAs, 5S rRNA, scR1 (SRP) and aborted tRNAs transcripts. Conversely, pre-tRNAs preferentially accessed Rrp44 directly. Both routes participated in degradation and maturation of RNAPI transcripts, with hand-over during processing. Rrp41 mutations blocked substrate passage through the channel to Rrp44 only for cytoplasmic mRNAs, supporting the predicted widening of the lumen in the Rrp6-associated, nuclear complex. Many exosome substrates exhibited clear preferences for a specific path to Rrp44. Other targets showed redundancy, possibly allowing the efficient handling of highly diverse RNA-protein complexes and RNA structures. Both threading and direct access routes involve the RNA helicase Mtr4. mRNAs that are predominately nuclear or cytoplasmic exosome substrates can be distinguished in vivo.

The RNA Exosome Channeling and Direct Access Conformations Have Distinct In Vivo Functions

The RNA exosome is a 3'-5' ribonuclease complex that is composed of nine core subunits and an essential catalytic subunit, Rrp44. Two distinct conformations of Rrp44 were revealed in previous structural studies, suggesting that Rrp44 may change its conformation to exert its function. In the channeling conformation, (Rrp44(ch)), RNA accesses the active site after traversing the central channel of the RNAexosome, whereas in the other conformation, (Rrp44(da)), RNA gains direct access to the active site. Here, we show that the Rrp44(da) exosome is important for nuclear function of the RNA exosome. Defects caused by disrupting the direct access conformation are distinct from those caused by channel-occluding mutations, indicating specific functions for each conformation. Our genetic analyses provide invivo evidence that the RNA exosome employs a direct-access route to recruit specific substrates, indicating that the RNA exosome uses alternative conformations to act on different RNA substrates.

Visualization of distinct substrate-recruitment pathways in the yeast exosome by EM

The eukaryotic exosome is a multi-subunit complex typically composed of a catalytically inactive core and the Rrp44 protein, which contains 3’ to 5’ exo- and endo-ribonuclease activities. RNA substrates have been shown to be recruited through the core to reach Rrp44's exoribonuclease (EXO) site. Using single particle electron microscopy and biochemical analysis, we provide visual evidence that two distinct substrate recruitment pathways exist. In the through-core route, channeling of the single stranded substrates from the core to Rrp44 induces a characteristic conformational change in Rrp44. In the alternative direct-access route, this conformational change does not take place and the RNA substrate is visualized to avoid the core and enter Rrp44's EXO site directly. Our results provide mechanistic explanations for several RNA processing scenarios by the eukaryotic exosome and indicate substrate specific modes of degradation by this complex.

On the role of thermal backbone fluctuations in myoglobin ligand gate dynamics

We construct an energy function that describes the crystallographic structure of sperm whale myoglobin backbone. As a model in our construction, we use the Protein Data Bank entry 1ABS that has been measured at liquid helium temperature. Consequently, the thermal B-factor fluctuations are very small, which is an advantage in our construction. The energy function that we utilize resembles that of the discrete nonlinear Schrödinger equation. Likewise, ours supports topological solitons as local minimum energy configurations. We describe the 1ABS backbone in terms of topological solitons with a precision that deviates from 1ABS by an average root-mean-square distance, which is less than the experimentally observed Debye-Waller B-factor fluctuation distance. We then subject the topological multi-soliton solution to extensive numerical heating and cooling experiments, over a very wide range of temperatures. We concentrate in particular to temperatures above 300 K and below the Θ-point unfolding temperature, which is around 348 K. We confirm that the behavior of the topological multi-soliton is fully consistent with Anfinsen's thermodynamic principle, up to very high temperatures. We observe that the structure responds to an increase of temperature consistently in a very similar manner. This enables us to characterize the onset of thermally induced conformational changes in terms of three distinct backbone ligand gates. One of the gates is made of the helix F and the helix E. The two other gates are chosen similarly, when open they provide a direct access route for a ligand to reach the heme. We find that out of the three gates we investigate, the one which is formed by helices B and G is the most sensitive to thermally induced conformational changes. Our approach provides a novel perspective to the important problem of ligand entry and exit.

An introduction to the semantics of intertextuality

Abstract This paper engages with intertextuality and proposes a cognitively-informed approach to it based on the notion of semantic intertextual frames, an online processing domain which regulates the construction of word-level intertextual links. The construction of these frames is discussed in terms of Evans' LCCM Theory (Theory of Lexical Concepts and Cognitive Models), and the distinction he draws between lexical concepts and cognitive models. Lexical concepts afford access to cognitive models via direct or indirect access routes. Word-level intertextual connections are explained based on the identification of the same lexical item, a cognitive synonym or a hyponym. In the first two cases a direct access route to the cognitive model is afforded by the lexical concepts, while in the latter this route is indirect. A number of examples drawn from literary texts are used to illustrate the model.

The processing of phonological, orthographical, and lexical information of Chinese characters in sentence contexts: An ERP study

In the current work, we aimed to study the processing of phonological, orthographical, and lexical information of Chinese characters in sentence contexts, as well as to provide further evidence for psychological models. In the experiment, we designed sentences with expected, homophonic, orthographically similar, synonymous, and control characters as endings, respectively. The results indicated that P200 might be related to the early extraction of phonological information. Moreover, it might also represent immediate semantic and orthographic lexical access. This suggested that there might be a dual-route in cognitive processing, where the direct access route and the phonologically mediated access route both exist and interact with each other. The increased N400 under the control condition suggested that both phonological and orthographical information would influence semantic integration in Chinese sentence comprehension. The two positive peaks of the late positive shift might represent the semantic monitoring, and orthographical retrieval and reanalysis processing, respectively. Under the orthographically similar condition, orthographical retrieval and reanalysis processing was more difficult in comparison with the other conditions, which suggested that there might be direct access from orthography to semantic representation in cognitive processing. In conclusion, it was shown that the direct access hypothesis or the dual-route hypothesis could better explain cognitive processing in the brain.

Can out‐of‐context musical sounds convey meaning? An ERP study on the processing of meaning in music

There has been much debate over whether music can convey extra-musical meaning. The experiments presented here investigated whether low level musical features, specifically the timbre of a sound, have a direct access route to meaningful representations. Short musical sounds with varying timbres were investigated with regard to their ability to elicit meaningful associations, and the neural mechanisms underlying the meaningful processing of sounds were compared to those underlying the semantic processing of words. Two EEG experiments were carried out, and N400 effects were found for sound and word targets following sound and word primes in a semantic relatedness judgment task. No N400 effects were found in a memory task. The results show that even short musical sounds outside of a musical context are capable of conveying meaning information, but that sounds require more elaborate processing than other kinds of meaningful stimuli.

Effect of Health Care Delivery Models on Melanoma Thickness and Stage in a University-Based Referral Center

To compare the effect of differing health care delivery models, specifically, gatekeeper (GK) vs direct access (DA) routes, on melanoma outcome as measured by tumor thickness and cancer stage at diagnosis. Retrospective medical record review of patients previously diagnosed as having cutaneous melanoma who were referred to a university-based clinic from January 1, 1996, through December 31, 2000. Stanford Pigmented Lesion and Cutaneous Melanoma Clinic, Stanford, Calif. Patients Two hundred thirty-four patients with primary melanoma stratified according to health care access route (GK or DA). Differences in Breslow thickness, American Joint Committee on Cancer stage, histologic features, patient delay in seeking medical attention, and physician delay in diagnosis (time between initial physician visit and diagnostic biopsy procedure). Of 234 patients, 168 (72%) were referred through the DA route and 66 (28%) through the GK route. A significant association was found between physician delay and access route; patients in the DA group underwent biopsy sooner (< or =3 months vs >3 months) than those in the GK group (P<.001). No significant difference was observed in stage at diagnosis (predominantly stage IA), proportion of nodular melanoma (DA 4% vs GK 2%), patient delay, or median tumor thickness between DA and GK routes (0.42 mm vs 0.50 mm, respectively). A trend toward a greater proportion of histologically ulcerated melanoma was observed in the DA group compared with the GK group (12% vs 5%, respectively; P = .06). This pilot study demonstrated no difference in outcome between GK and DA routes as measured by melanoma thickness and stage, although patients in the DA group underwent diagnostic biopsy sooner than those in the GK group. The potential effect of health care models on melanoma outcomes merits further study.

From Speech Perception to Morphology: Affix Ordering Revisited

From Speech Perception to Morphology: Affix Ordering Revisited Jennifer Hay This article presents corpus and experimental evidence in support of a parsability-based account of affix ordering in English: an affix that tends to be easily parsed out during speech perception should not occur inside an affix that does not. This generalization holds both at the affix level and the word level. At the affix level, this maxim, when combined with an understanding of the role of frequency and phonotactics in morphological processing, can account for the patterns generally attributed to level ordering. At the word level, it can explain the so-called dual-level behavior of some affixes—an affix may resist attaching to a complex word that is highly decomposable but be acceptable when it attaches to a comparable complex word that favors the direct access route in speech perception. Only a parsing account can afford this set of phenomena a unified explanation.* Introduction One of the most debated problems of English morphology is that of stacking restrictions amongst derivational affixes. Of the many potential combinations of affixes, only a very small proportion are actually attested. Many attempts to account for apparent restrictions on affix ordering have invoked some form of the affix-ordering generalization (Siegel 1979). The basic claim of the affix-ordering generalization is that affixes can be divided into two sets: level 1 and level 2. Level 1 affixation occurs prior to level 2 affixation, and so no level 1 affix can attach outside of any level 2 affix (hence *-ness-ic, *-less-ity, and so on). I argue here that early accounts of affix ordering were overly restrictive and drew the line at the wrong level of abstraction. But more recent work, which has discarded the idea that there are any restrictions on ordering (beyond selectional restrictions), misses a number of important generalizations. Many facts about English stacking restrictions can be predicted if we reduce the problem to one of parsability. This can capture not only the range of generalizations about English stacking restrictions but also a large number of systematic, word-based exceptions to these generalizations. In a paper widely cited as responsible for disproving the affix-ordering generalization, Fabb (1988) argues against a stratificational approach to affix ordering. He demonstrates that the affix-ordering generalization fails to rule out a large number of affix combinations, which nonetheless do not occur. He argues that affix ordering is constrained only by selectional restrictions: all but a few non-occurring pairs of suffixes can be ruled out solely by a combination of selectional restrictions, of which one of the most extensive in its effects is a restriction against attachment to an already suffixed word. (Fabb 1988:538) One of Fabb’s observations, then, is that there seem to be a large number of affixes that do not attach to already affixed words. This observation, I argue, holds the key to understanding restrictions on affix ordering in English. Many affixes are sensitive to internal structure in potential bases. While some affixes basically tolerate no internal structure, others will tolerate structure to some minimum degree. The degree of internal [End Page 527] structure tolerated by an affix is not determined by selectional restrictions, but, rather, by how much structure that affix itself creates. Phrased in terms of processing, an affix that can be easily parsed out should not occur inside an affix that cannot. Details on how to operationalize the notion of ease of parsing are provided below. I demonstrate that this maxim, when combined with an understanding of the role of frequency and phonology in morphological processing, accounts neatly for restrictions on affix ordering in English. We do not find any cases in which an affix attaches only to forms that are maximally decomposable, and not to forms that are relatively opaque, though there are large numbers of cases where the opposite holds true. The range of results put forward here cannot be accounted for by the classical affix-ordering account, nor by any account involving selectional restrictions. The account presented here takes as its starting point some fundamentals of speech perception. Before turning to affix ordering, then, I will outline some basic assumptions about...

Colonisation par des ammonites méridionales, des mers subboréales kimméridgiennes du Yorkshire (Angleterre)

During the Kimmeridgian (Mutabilis, Eudoxus and Autissiodorensis Zones), southern ammonites from the submediterranean province, colonized the subboreal seas of Yorkshire (England) on several separate occasions. They are aspidoceratids, Sutneria and later Tolvericeras.. Besides the classic communication route via the northern Paris Basin and Dorset, another more direct access route, from the Swabian and Franconian Alb through the Hesse Strait, is now recognised. These colonisations indicate periods of high sea level and increased oceanic circulation. A rise in temperature and/or a nutrient enrichment may also have been involved. At the top of the Elegans Zone, the arrival of isolated Gravesia,, typical of the Franco-German biome, enables correlations between the Gravesia and Elegans/Scitulus Zones of the submediterranean and subboreal provinces respectively. Yorkshire is the most northerly occurrence of all these southern ammonites.

The antimalarial action of desferal involves a direct access route to erythrocytic (Plasmodium falciparum) parasites.

We designed the N-methylanthranilic-desferrioxamine (MA-DFO) as a fluorescent iron (III) chelator with improved membrane permeation properties. Upon binding of iron (III), MA-DFO fluorescence is quenched, thus allowing traceability of drug-iron (III) interactions. MA-DFO is well tolerated by mammalian cells in culture. Its antimalarial activity is pronounced: IC50 values on in vitro (24-h) growth of Plasmodium falciparum were 3 +/- 1 microM for MA-DFO compared with 30 +/- 8 for DFO. The onset of growth inhibition of rings or trophozoites occurs 2-4 h after exposure to 13 microM MA-DFO. This effect is commensurate with MA-DFO permeation into infected cells. In a 24-h exposure to MA-DFO or DFO, trophozoites take up either compound to approximately 10% of the external concentration, rings to 5%, and noninfected cells to < 1%. Red cells encapsulated with millimolar concentrations of DFO or MA-DFO fully support parasite invasion and growth. We conclude that extracellular MA-DFO and DFO gain selective access into parasites by bypassing the host. The rate-limiting step is permeation through the parasite membrane, which MA-DFO accomplishes faster than DFO, in accordance with its higher hydrophobicity. These views are consistent with the proposed duct, which apparently provides parasitized cells with a window to the external medium.

Re-education of a deep dyslexic patient: Rationale of the method and results

Abstract A report on the re-education of a deep dyslexic patient with fluent speech is presented. Results of extensive pre-therapeutic testing suggested that the patient was using a direct access route to meaning and that the grapheme-to-phoneme conversion rules were impaired. To re-educate the reading abilities of our patient, we chose a reorganisational rationale, that is, we tried to reorganise the impaired grapheme-to-phoneme process by using spared lexical knowledge as a relay between graphemes and their pronunciation. Our therapy was divided into three main stages: 1. single grapheme-reading reconstruction by using word codes as an intermediary 2. complex grapheme-reading reconstruction by using a homophonic word code, and 3. learning graphemic contextual rules. After a year of intensive practice to automate the new strategies, the patient was able to read slowly and correctly.