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<input type="email" aria-label="Recipient Email Address" placeholder="email@example.com" id="email-to" class="email-to" pattern="^[a-zA-Z0-9_.+-]+@[a-zA-Z0-9-]+\.[a-zA-Z0-9-.]+$" maxlength="100" required>
</div>
<div class="action-panel-control-wrap">
<label for="email-from" class="action-panel-label">
From:
</label>
<input type="email" aria-label="Sender Email Address" placeholder="email@example.com" id="email-from" class="email-from" pattern="^[a-zA-Z0-9_.+-]+@[a-zA-Z0-9-]+\.[a-zA-Z0-9-.]+$" maxlength="256">
</div>
<div class="action-panel-control-wrap">
<label for="email-citation-format" class="action-panel-label">
Format:
</label>
<select id="email-citation-format" name="citation-format" class="action-panel-selector email-citation-format">
<option selected="selected" value="summary">Summary</option>
<option value="summary-text">Summary (text)</option>
<option value="abstract">Abstract</option>
<option value="abstract-text">Abstract (text)</option>
</select>
</div>
<div class="include-supplemental-container">
<input type="checkbox" aria-label="Include MeSH and other data" name="include-supplemental" id="email-include-supplemental" class="email-include-supplemental">
<label for="email-include-supplemental" class="email-include-supplemental-label">MeSH and other data</label>
</div>
<div class="form-field recaptcha ">
<div class="g-recaptcha" id="id-recaptcha" data-sitekey="6LfsWHMdAAAAAClKbtOpjQ2pMjgsGxvv7NdZW9uI"></div>
</div>
<div id="captcha-error-message" class="usa-input-error-message captcha-validation-message" role="alert"></div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Sending..."
data-ga-category="save_share"
data-ga-action="email"
data-ga-label="send">
Send email
</button>
<button class="action-panel-cancel"
aria-label="Close 'Email citations' panel"
ref="linksrc=close_email_panel"
aria-controls="email-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="email"
data-ga-label="cancel">
Cancel
</button>
</div>
<input type="hidden" name="email-search-details" value="" />
<input type="hidden" name="email-search-details-hash" value="0e42663a6c3bd85498fcb88798998fed7bfdc45d457db35281e41afe13cc0524" />
</form>
</div>
</div>
<div id="collections-action-panel"
class="collections-action-panel action-panel in-progress-dots-panel"
aria-hidden="true"
data-collections-open-panel-enabled="false"
data-collections-open-panel-url-hash="#open-collections-panel">
<div class="inner-wrap">
<h3 class="action-panel-heading">
Add to Collections
</h3>
<form id="collections-action-panel-form"
class="collections-action-panel-form action-panel-content action-form action-panel-smaller-selectors"
data-existing-collections-url="/list-existing-collections/"
data-add-to-existing-collection-url="/add-to-existing-collection/"
data-create-and-add-to-new-collection-url="/create-and-add-to-new-collection/"
data-get-article-ids-by-search-url="/get-article-ids-by-search/"
data-myncbi-max-collection-name-length="100"
data-add-to-collection-max-amount="1000"
data-collections-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/">
<input type="hidden" name="csrfmiddlewaretoken" value="z9oV9hxNTxjnqLG9txxsvCydscPertdAQW80M709t86RkcMZ6QauZK4R0gxqk4LU">
<div class="choice-group" role="radiogroup">
<ul class="radio-group-items">
<li>
<input type="radio"
id="collections-action-panel-new"
class="collections-new"
name="collections"
value="new"
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="collections_radio_new">
<label for="collections-action-panel-new">Create a new collection</label>
</li>
<li>
<input type="radio"
id="collections-action-panel-existing"
class="collections-existing"
name="collections"
value="existing"
checked="true"
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="collections_radio_existing">
<label for="collections-action-panel-existing">Add to an existing collection</label>
</li>
</ul>
</div>
<div class="controls-wrapper">
<div class="action-panel-control-wrap new-collections-controls">
<label for="collections-action-panel-add-to-new" class="action-panel-label required-field-asterisk">
Name your collection:
</label>
<input
type="text"
name="add-to-new-collection"
id="collections-action-panel-add-to-new"
class="collections-action-add-to-new"
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/"
maxlength="100"
data-ga-category="save_share"
data-ga-action="create_collection"
data-ga-label="non_favorties_collection">
<div class="collections-new-name-too-long usa-input-error-message selection-validation-message">
Name must be less than 100 characters
</div>
</div>
<div class="action-panel-control-wrap existing-collections-controls">
<label for="collections-action-panel-add-to-existing" class="action-panel-label">
Choose a collection:
</label>
<select id="collections-action-panel-add-to-existing"
class="action-panel-selector collections-action-add-to-existing"
data-ga-category="save_share"
data-ga-action="select_collection"
data-ga-label="($('#collections-action-add-to-existing').val() === 'Favorites') ? 'Favorites' : 'non_favorites_collection'">
</select>
<div class="collections-retry-load-on-error usa-input-error-message selection-validation-message">
Unable to load your collection due to an error<br>
<a href="#">Please try again</a>
</div>
</div>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Adding..."
data-pinger-ignore
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="add">
Add
</button>
<button class="action-panel-cancel"
aria-label="Close 'Add to Collections' panel"
ref="linksrc=close_collections_panel"
aria-controls="collections-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="bibliography-action-panel"
class="bibliography-action-panel action-panel in-progress-dots-panel"
aria-hidden="true"
data-bibliography-open-panel-enabled="false"
data-bibliography-open-panel-url-hash="#open-bibliography-panel">
<div class="inner-wrap">
<h3 class="action-panel-heading">
Add to My Bibliography
</h3>
<form id="bibliography-action-panel-form"
class="bibliography-action-panel-form action-panel-content action-form action-panel-smaller-selectors"
data-add-to-bibliography-max-amount="100"
data-add-to-bibliography-batch-size="10"
data-bibliography-delegates-url="/list-bibliography-delegates/"
data-add-to-bibliography-url="/add-to-bibliography/"
data-get-article-ids-by-search-url="/get-article-ids-by-search/"
data-mybib-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/mybibliography/">
<input type="hidden" name="csrfmiddlewaretoken" value="z9oV9hxNTxjnqLG9txxsvCydscPertdAQW80M709t86RkcMZ6QauZK4R0gxqk4LU">
<div class="action-panel-control-wrap bibliographies-controls">
<div class="choice-group">
<ul class="bibliographies-action-add radio-group-items">
<li>
<input name="bibliography" id="my-bibliography" class="my-bibliography" type="radio" checked/>
<label for="my-bibliography">My Bibliography</label>
</li>
</ul>
</div>
</div>
<div class="bibliographies-retry-load-on-error usa-input-error-message selection-validation-message">
Unable to load your delegates due to an error<br>
<a href="#">Please try again</a>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Adding..."
data-pinger-ignore>
Add
</button>
<button class="action-panel-cancel"
aria-label="Close 'Add to bibliography' panel"
ref="linksrc=close_bibliography_panel"
aria-controls="bibliography-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="mybib"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="saved-search-action-panel" class="saved-search-action-panel action-panel " aria-hidden="true"
data-saved-search-open-panel-enabled="false"
data-saved-search-open-panel-url-hash="#open-saved-search-panel">
<div class="inner-wrap">
<h2 class="action-panel-heading">
Your saved search
</h2>
<form id="saved-search-action-panel-form"
class="saved-search-action-panel-form action-panel-content action-form"
data-create-saved-search-url="/create-saved-search/"
data-try-search-terms-url="/try-search-term/"
data-saved-search-root-url="https://www.ncbi.nlm.nih.gov/myncbi/searches/">
<input type="hidden" name="csrfmiddlewaretoken" value="z9oV9hxNTxjnqLG9txxsvCydscPertdAQW80M709t86RkcMZ6QauZK4R0gxqk4LU">
<div class="action-panel-control-wrap">
<label for="saved-search-name" class="action-panel-label saved-search-name-label required-field-asterisk">
Name of saved search:
</label>
<input maxlength="200"
type="text"
name="saved-search-name"
id="saved-search-name"
class="saved-search-name"
value=""
required
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/">
</div>
<div class="action-panel-control-wrap">
<label for="saved-search-term" class="action-panel-label required-field-asterisk">
Search terms:
</label>
<textarea name="saved-search-term" id="saved-search-term" class="saved-search-term" required></textarea>
</div>
<div class="test-search-term-wrap">
<a href="#" class="try-search-term">Test search terms</a>
</div>
<div class="choice-group action-panel-extra-margin-top">
<span class="action-panel-label" id="fieldset-label">
Would you like email updates of new search results?
</span>
<fieldset id="saved-search-alert" aria-describedby="fieldset-label">
<legend class="usa-sr-only">Saved Search Alert Radio Buttons</legend>
<ul class="radio-group-items">
<li>
<input type="radio" id="saved-search-alert-yes" class="saved-search-alert-yes" name="saved-search-alert" value="yes" checked>
<label for="saved-search-alert-yes" class="action-panel-label">Yes</label>
</li>
<li>
<input aria-label="No radio input" type="radio" id="saved-search-alert-no" class="saved-search-alert-no" name="saved-search-alert" value="no">
<label for="saved-search-alert-no" class="action-panel-label">No</label>
</li>
</ul>
</fieldset>
</div>
<div class="alert-schedule-wrap">
<div class="action-panel-control-wrap">
<label class="action-panel-label">
Email:
</label>
<span aria-label="Email address" id="saved-search-email" class="action-panel-label"><span class="action-panel-label-bold"></span> (<a class="myncbi-account-settings" href="https://www.ncbi.nlm.nih.gov/account/settings/">change</a>)</span>
</div>
<div class="action-panel-control-wrap action-panel-extra-margin-top">
<label for="saved-search-frequency" class="action-panel-label">
Frequency:
</label>
<select id="saved-search-frequency" class="no-border-panel-selector saved-search-frequency">
<option value="monthly">Monthly</option>
<option value="weekly">Weekly</option>
<option value="daily">Daily</option>
</select>
</div>
<div class="action-panel-control-wrap saved-search-monthly-additional">
<label for="saved-search-monthly-on-day" class="action-panel-label">
Which day?
</label>
<select id="saved-search-monthly-on-day" class="no-border-panel-selector">
<option value="Sunday">The first Sunday</option>
<option value="Monday">The first Monday</option>
<option value="Tuesday">The first Tuesday</option>
<option value="Wednesday">The first Wednesday</option>
<option value="Thursday">The first Thursday</option>
<option value="Friday">The first Friday</option>
<option value="Saturday">The first Saturday</option>
<option value="day">The first day</option>
<option value="weekday">The first weekday</option>
</select>
</div>
<div class="action-panel-control-wrap saved-search-weekly-additional">
<label for="saved-search-weekly-on-day" class="action-panel-label">
Which day?
</label>
<select id="saved-search-weekly-on-day" class="no-border-panel-selector saved-search-weekly-on-day">
<option value="Sunday">Sunday</option>
<option value="Monday">Monday</option>
<option value="Tuesday">Tuesday</option>
<option value="Wednesday">Wednesday</option>
<option value="Thursday">Thursday</option>
<option value="Friday">Friday</option>
<option value="Saturday">Saturday</option>
</select>
</div>
<div class="action-panel-control-wrap">
<label for="saved-search-report" class="action-panel-label">
Report format:
</label>
<select id="saved-search-report" class="no-border-panel-selector saved-search-report">
<option value="DocSum">Summary</option>
<option value="DocSumText">Summary (text)</option>
<option value="Abstract">Abstract</option>
<option value="AbstractText">Abstract (text)</option>
<option value="MEDLINE">PubMed</option>
</select>
</div>
<div class="action-panel-control-wrap">
<label for="saved-search-amount" class="action-panel-label">
Send at most:
</label>
<select id="saved-search-amount" class="no-border-panel-selector saved-search-amount">
<option value="1">1 item</option>
<option value="5" selected>5 items</option>
<option value="10">10 items</option>
<option value="20">20 items</option>
<option value="50">50 items</option>
<option value="100">100 items</option>
<option value="200">200 items</option>
</select>
</div>
<div>
<input type="checkbox" id="saved-search-send-if-no-result" class="saved-search-send-if-no-result" name="saved-search-send-if-no-result">
<label for="saved-search-send-if-no-result" class="action-panel-label smaller-checkbox">
Send even when there aren't any new results
</label>
</div>
<div class="action-panel-control-wrap option-text-in-email-wrap">
<label for="saved-search-email-text" class="action-panel-label">
Optional text in email:
</label>
<textarea name="saved-search-email-text"
id="saved-search-email-text"
class="saved-search-email-text"></textarea>
</div>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Saving..."
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="save">
Save
</button>
<button class="action-panel-cancel"
aria-label="Close 'Your saved search' panel"
ref="linksrc=close_saved_search_panel"
aria-controls="saved-search-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="citation-manager-action-panel" class="citation-manager-action-panel action-panel" aria-hidden="true">
<div class="inner-wrap">
<h2 class="action-panel-heading">
Create a file for external citation management software
</h2>
<form id="citation-manager-action-panel-form"
class="action-panel-content action-form"
action="/results-export-ids/"
data-by-search-action="/results-export-search-data/"
data-by-ids-action="/results-export-ids/"
method="post"
data-by-search-method="post"
data-by-ids-method="post">
<input type="hidden" name="csrfmiddlewaretoken" value="z9oV9hxNTxjnqLG9txxsvCydscPertdAQW80M709t86RkcMZ6QauZK4R0gxqk4LU">
<input name="results-format" type="hidden" value="pubmed"/>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Sending..."
data-ga-category="save_share"
data-ga-action="citation_manager"
data-ga-label="save">
Create file
</button>
<button class="action-panel-cancel"
aria-label="Close 'Send citations to citation manager' panel"
ref="linksrc=close_citation_manager_panel"
aria-controls="citation-manager-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="citation_manager"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="rss-action-panel" class="rss-action-panel action-panel " aria-hidden="true">
<div class="inner-wrap">
<h2 class="action-panel-heading">
Your RSS Feed
</h2>
<form id="rss-action-panel-form"
class="rss-action-panel-form action-panel-content action-form"
data-create-rss-feed-url="/create-rss-feed-url/"
data-search-form-term-value="">
<input type="hidden" name="csrfmiddlewaretoken" value="z9oV9hxNTxjnqLG9txxsvCydscPertdAQW80M709t86RkcMZ6QauZK4R0gxqk4LU">
<div class="action-panel-control-wrap">
<label for="rss-name" class="action-panel-label required-field-asterisk">
Name of RSS Feed:
</label>
<input maxlength="200"
placeholder="Name"
type="text"
name="rss-name"
id="rss-name"
class="rss-name"
value=''
required
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/">
</div>
<div class="rss-limit-wrap">
<div class="action-panel-control-wrap action-panel-extra-margin-top">
<label for="rss-limit" class="action-panel-label">
Number of items displayed:
</label>
<select id="rss-limit" class="no-border-panel-selector rss-limit">
<option value="5">5</option>
<option value="10">10</option>
<option value="15" selected="selected">15</option>
<option value="20">20</option>
<option value="50">50</option>
<option value="100">100</option>
</select>
</div>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Creating..."
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="save">
Create RSS
</button>
<button class="action-panel-cancel"
aria-label="Close 'Your RSS' panel"
ref="linksrc=close_rss_panel"
aria-controls="rss-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="cancel">
Cancel
</button>
</div>
<div class="action-panel-control-wrap rss-link-copy-wrap">
<label for="rss-link" class="usa-sr-only">RSS Link</label>
<input placeholder="Your RSS Feed Link" type="text" name="rss-link" id="rss-link" class="rss-link" title="RSS Link">
<button
type="button"
disabled
class="rss-link-copy-button disabled"
data-ga-category="save_share"
data-ga-action="rss"
data-ga-label="copy">
Copy
</button>
</div>
</form>
</div>
</div>
</div>
</div>
<div class="article-page" id="article-page" data-article-pmid="16885028">
<aside class="page-sidebar">
<div class="inner-wrap">
<div class="full-text-links">
<div class="full-view">
<h3 class="title">
Full text links
</h3>
<div class="full-text-links-list">
<a class="link-item
dialog-focus"
href="https://linkinghub.elsevier.com/retrieve/pii/S1097-2765(06)00413-8"
target="_blank"
rel="noopener"
ref="linksrc=fulltextorjournal_fulltext&amp;is_pmc=False&amp;PrId=3048&amp;itool=Abstract-def&amp;log$=linkouticon&amp;uid=16885028&amp;db=pubmed&amp;nlmid=9802571"
title="See full text options at Elsevier Science"
data-ga-category="full_text"
data-ga-action="Elsevier Science"
data-ga-label="16885028"
><img src="https://cdn.ncbi.nlm.nih.gov/corehtml/query/egifs/https:--linkinghub.elsevier.com-ihub-images-celloa.png" alt="Elsevier Science full text link"><span class="text">
Elsevier Science
</span></a><a class="link-item
pmc
"
href="https://pmc.ncbi.nlm.nih.gov/articles/pmid/16885028/"
target="_blank"
rel="noopener"
ref="linksrc=fulltextorjournal_fulltext&amp;is_pmc=True&amp;PrId=3494&amp;itool=Abstract-def&amp;log$=linkouticon&amp;uid=16885028&amp;db=pubmed&amp;nlmid=9802571"
title="Free full text at PubMed Central"
data-ga-category="full_text"
data-ga-action="PMC"
data-ga-label="16885028"
><span class="text">
Free PMC article
</span></a>
</div>
</div>
<div class="short-view">
<a href="#" class="full-text-links-button full-text-links-dialog-trigger">
Full text links
</a>
</div>
</div>
<div class="actions-buttons sidebar"><h3 class="title">Actions</h3><div class="inner-wrap"><button class="citation-button citation-dialog-trigger"
aria-label="Open dialog with citation text in different styles"
data-ga-category="save_share"
data-ga-action="cite"
data-ga-label="open"
data-all-citations-url="/16885028/citations/"
data-citation-style="nlm"
data-pubmed-format-link="/16885028/export/"><span class="button-label">Cite</span></button><link type="text/css" href="ncbi-overlay-block/src/overlay-block.css"><div class="collections-button-container" data-article-id="16885028" data-article-db="pubmed"><button class="collections-button collections-dialog-trigger"
aria-label="Save article in MyNCBI collections."
data-ga-category="collections_button"
data-ga-action="click"
data-ga-label="collections_button"
data-collections-open-dialog-enabled="false"
data-collections-open-dialog-url="https://account.ncbi.nlm.nih.gov/?back_url=https%3A%2F%2Fpubmed.ncbi.nlm.nih.gov%2F16885028%2F%23open-collections-dialog"
data-in-collections="false"><span class="button-label">Collections</span></button><div class="overlay" role="dialog"><div id="collections-action-dialog"
class="dialog collections-dialog"
aria-hidden="true"><div class="title">Add to Collections</div><div class="collections-action-panel action-panel"><form id="collections-action-dialog-form"
class="collections-action-panel-form action-panel-content action-form action-panel-smaller-selectors"
data-existing-collections-url="/list-existing-collections/"
data-add-to-existing-collection-url="/add-to-existing-collection/"
data-create-and-add-to-new-collection-url="/create-and-add-to-new-collection/"
data-myncbi-max-collection-name-length="100"
data-collections-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/"><input type="hidden" name="csrfmiddlewaretoken" value="z9oV9hxNTxjnqLG9txxsvCydscPertdAQW80M709t86RkcMZ6QauZK4R0gxqk4LU"><div class="choice-group" role="radiogroup"><ul class="radio-group-items"><li><input type="radio"
id="collections-action-dialog-new"
class="collections-new"
name="collections"
value="new"
data-ga-category="collections_button"
data-ga-action="click"
data-ga-label="collections_radio_new"><label for="collections-action-dialog-new">Create a new collection</label></li><li><input type="radio"
id="collections-action-dialog-existing"
class="collections-existing"
name="collections"
value="existing"
checked="true"
data-ga-category="collections_button"
data-ga-action="click"
data-ga-label="collections_radio_existing"><label for="collections-action-dialog-existing">Add to an existing collection</label></li></ul></div><div class="controls-wrapper"><div class="action-panel-control-wrap new-collections-controls"><label for="collections-action-dialog-add-to-new" class="action-panel-label required-field-asterisk">
Name your collection:
</label><input
type="text"
name="add-to-new-collection"
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The EF-G-like GTPase Snu114p regulates spliceosome dynamics mediated by Brr2p, a DExD/H box ATPase
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The EF-G-like GTPase Snu114p regulates spliceosome dynamics mediated by Brr2p, a DExD/H box ATPase
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Binding of a pre-mRNA substrate triggers spliceosome activation, whereas the release of the mRNA product triggers spliceosome disassembly. The mechanisms that underlie the regulation of these rearrangements remain unclear. We find evidence that the GTPase Snu114p mediates the regulation of spliceosome activation and disassembly. Specifically, both unwinding of U4/U6, required for spliceosome activation, and disassembly of the postsplicing U2/U6.U5.intron complex are repressed by Snu114p bound to GDP and derepressed by Snu114p bound to GTP or nonhydrolyzable GTP analogs. Further, similar to U4/U6 unwinding, spliceosome disassembly requires the DExD/H box ATPase Brr2p. Together, our data define a common mechanism for regulating and executing spliceosome activation and disassembly. Although sequence similarity with EF-G suggests Snu114p functions as a molecular motor, our findings indicate that Snu114p functions as a classic regulatory G protein. We propose that Snu114p serves as a signal-dependent switch that transduces signals to Brr2p to control spliceosome dynamics.
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<p> Figure 1 </p>
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<div class="figure-caption-contents"><p> Purification of in vivo Assembled… </p></div>
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<p> Figure 1 </p>
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<div class="figure-caption-contents"><p> Purification of in vivo Assembled Spliceosomes Poised for Disassembly (A) Prp43p- and Prp22p-associated… </p></div>
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Figure 1
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<div class="figure-caption-contents">Purification of in vivo Assembled Spliceosomes Poised for Disassembly (A) Prp43p- and Prp22p-associated spliceosomes, affinity-purified from yJPS797 or yJPS773 using a tandem affinity purification (TAP) tag (Rigaut et al., 1999), are enriched for U2, U5 and U6. RNA was extracted from lysate (L) or purified spliceosomes (P); 1% or 100%, respectively, was analyzed by primer extension (top). The percent yield for each snRNA is shown for Prp43p-(black) and Prp22p-(grey) associated spliceosomes (bottom). (B) Prp43p-associated spliceosomes are enriched specifically for excised intron, whereas Prp22p-associated spliceosomes are enriched for excised intron and ligated exons. RNA was extracted from lysate (L) or purified spliceosomes (P); 0.4% or 100%, respectively, was probed by Northern for the U3A pre-snoRNA, snoRNA and excised intron, indicated from top to bottom on the left. (C) In Prp43p-associated spliceosomes, the U3A intron is branched. RNA extracted from spliceosomes was incubated without (-) or with (+) debranchase (Dbr1p), as described (Khalid et al., 2005) for 30 min at 22°C, and probed by Northern for the branched and debranched introns, depicted from top to bottom on the left. (D) In Prp43p-associated spliceosomes, U2 crosslinks with U6. After UV-irradiating spliceosomes, RNA was extracted and probed sequentially by Northern for U6, U2 and U5, as indicated below the blots. The crosslinked species is indicated as U2/U6.</div>
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<p> Figure 2 </p>
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<div class="figure-caption-contents"><p> Prp43p-associated Spliceosomes Disassemble in an… </p></div>
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<p> Figure 2 </p>
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<div class="figure-caption-contents"><p> Prp43p-associated Spliceosomes Disassemble in an ATP- and Prp43p-Dependent Manner (A) A Prp43p-associated U2/U6•U5•intron… </p></div>
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Figure 2
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<div class="figure-caption-contents">Prp43p-associated Spliceosomes Disassemble in an ATP- and Prp43p-Dependent Manner (A) A Prp43p-associated U2/U6•U5•intron complex disassembles rapidly in the presence of ATP. Complexes were purified from yJPS797 and incubated at 4°C in the presence (+) or absence (-) of ATP. At the indicated times, samples from the reactions were quenched and then analyzed by native gel followed by serial Northern probing. The migration of the U2/U6•U5•intron complex; the free U3A intron, and the free U6, U2 and U5 snRNAs are indicated on the left. (B) Disassembly of the U2/U6•U5•intron complex requires hydrolyzable ATP. Reactions were incubated at 4°C for 60 s and analyzed as in (A). (C) Disassembly of the U2/U6•U5•intron complex is promoted preferentially by ATP. Complexes were incubated at 4°C and analyzed as in (A). (D) ATP does not promote disassembly of Prp22p-associated spliceosomes. Prp43p-associated U2/U6•U5•intron complexes, purified from yJPS797, and Prp22p-associated U2/U6•U5•intron•mRNA complexes, purified from yJPS773, were incubated and analyzed as in (B). (E) Disassembly of Prp43p-associated spliceosomes is inhibited by the <i>prp43-Q423N</i> mutation but not by the <i>prp22-G810A</i> mutation. Spliceosomes were purified from wild-type <i>PRP43</i> (yJPS797) or <i>PRP22</i> (yJPS1049) and mutant <i>prp43-Q423N</i> (yJPS798) or <i>prp22-G810A</i> (yJPS1052) and then incubated and analyzed as in (B).</div>
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<strong class="figure-label">
<p> Figure 3 </p>
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<div class="figure-caption-contents"><p> Spliceosome Disassembly Requires the DExD/H-box… </p></div>
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<strong class="figure-label">
<p> Figure 3 </p>
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<div class="figure-caption-contents"><p> Spliceosome Disassembly Requires the DExD/H-box ATPase Brr2p (A) Disassembly of Prp43p-associated spliceosomes is… </p></div>
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Figure 3
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<div class="figure-caption-contents">Spliceosome Disassembly Requires the DExD/H-box ATPase Brr2p (A) Disassembly of Prp43p-associated spliceosomes is inhibited by <i>brr2</i> mutations. Spliceosomes were purified from the wild-type strain <i>BRR2</i> (yJPS999) or the mutant strains <i>brr2-1</i> (yJPS1000) and <i>brr2-R1107A</i> (yJPS1001). Spliceosomes were incubated and analyzed as in Figure 2B. (B) As for <i>brr2-1</i>, the mutant <i>brr2-R1107A</i> is cold sensitive. Cells were diluted from liquid culture and grown on rich media for 2 days at 30°C or 6 days at 15°C. (C) In <i>brr2</i> mutants, the excised lariat intron from U3A accumulates. Wild-type <i>BRR2</i> (yJPS999) or the mutants <i>brr2-1</i> (yJPS1000) and <i>brr2-R1107A</i> (yJPS1001) were grown in rich liquid media at 30°C and shifted to 15°C for 4 h. RNA was analyzed by Northern as in Figure 1B.</div>
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<strong class="figure-label">
<p> Figure 4 </p>
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<div class="figure-caption-contents"><p> Spliceosome Disassembly is Promoted and… </p></div>
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<p> Figure 4 </p>
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<div class="figure-caption-contents"><p> Spliceosome Disassembly is Promoted and Regulated by the GTPase Snu114p (A) Disassembly of… </p></div>
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Figure 4
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<div class="figure-caption-contents">Spliceosome Disassembly is Promoted and Regulated by the GTPase Snu114p (A) Disassembly of Prp43p-associated spliceosomes is inhibited by <i>snu114</i> mutants. Spliceosomes were purified from the wild-type strain <i>SNU114</i> (yJPS1053) or the mutant strains <i>snu114-14</i> (yJPS1120) and <i>snu114-60</i> (yJPS1056). Spliceosomes were incubated and analyzed as in Figure 2E. (B) <i>prp43</i> and <i>snu114</i> mutants genetically interact. yTB135 was cotransformed with plasmids encoding <i>PRP43</i> (pJPS1346) or <i>prp43-Q423N</i> (pJPS1347) and <i>SNU114</i> (pTB106), <i>snu114-14</i> (pTB108) or <i>snu114-60</i> (pTB113), grown in liquid media, diluted and grown on solid media containing 5-fluoroorotic acid for 5 days at 30°C. (C) Spliceosome disassembly is repressed specifically by GDP and derepressed specifically by GTP. Prp43p-associated spliceosomes, purified from a wild-type strain (yJPS797), were incubated at 4°C for 60 s and analyzed as in Figure 2B. (D) Spliceosome disassembly is regulated by Snu114p. Prp43p-associated spliceosomes were purified from a wild-type strain (yJPS1053) or a <i>snu114-D271N</i> mutant strain (yJPS1054) that appears to switch the specificity of Snu114p from guanine to xanthine (Bartels et al., 2003). Spliceosomes were incubated and analyzed as in Figure 2B. ATP, GDP and XDP concentrations were 2 mM; GTP and XTP concentrations were 0.5 mM. (E) Spliceosome disassembly does not require GTP hydrolysis. Prp43p-associated spliceosomes, purified from wild-type <i>SNU114</i> (yJPS1053) or mutant <i>snu114-D271N</i> (yJPS1054), were incubated at 4°C for 60 s and analyzed as in Figure 2B.</div>
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<strong class="figure-label">
<p> Figure 5 </p>
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<div class="figure-caption-contents"><p> U4/U6 Unwinding is Regulated by… </p></div>
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<p> Figure 5 </p>
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<div class="figure-caption-contents"><p> U4/U6 Unwinding is Regulated by Snu114p (A) Purification of U4/U6•U5 snRNPs associated with… </p></div>
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Figure 5
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<div class="figure-caption-contents">U4/U6 Unwinding is Regulated by Snu114p (A) Purification of U4/U6•U5 snRNPs associated with Prp28p. U4/U6•U5 snRNPs were affinity purified from lysates of yJPS1004 after glycerol gradient fractionation. By primer extension, RNA was analyzed from lysate (L) and pooled glycerol gradient fractions (F) and after immunoprecipitation from the supernatant (S) and the purified snRNP (P). (B) U4/U6 unwinding is repressed specifically by GDP and derepressed specifically by GTP. Prp28p-associated U4/U6•U5 snRNPs were incubated at 4°C and quenched after 60 s. RNA was extracted at 4°C, resolved on a native RNA gel and analyzed by Northern, probing simultaneously for U4 and U6. The migration of base paired U4/U6 and free U4 and U6 are indicated to the left. (C) U4/U6 unwinding is regulated by Snu114p. Prp28p-associated U4/U6•U5 snRNPs were purified from wild-type <i>SNU114</i> (yJPS1108) or mutant <i>snu114-D271N</i> (yJPS1111). Nucleotide concentrations were as in Figure 4D. snRNPs were incubated and analyzed as in (B). (D) U4/U6 unwinding does not require GTP hydrolysis. Prp28p-associated U4/U6•U5 snRNPs purified from wild-type <i>SNU114</i> (yJPS1108) or mutant <i>snu114-D271N</i> (yJPS1111) were incubated and analyzed as in (B). (E) U4/U6 unwinding requires the first Sec63 domain of Brr2p. Prp28p-associated U4/U6•U5 snRNPs were purified from a wild-type <i>BRR2</i> (yJPS1115) or mutant <i>brr2-R1107A</i> (yJPS1117) strain and incubated and analyzed as in (B).</div>
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<p> Figure 6 </p>
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<div class="figure-caption-contents"><p> A Role for Snu114p in… </p></div>
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<p> Figure 6 </p>
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<div class="figure-caption-contents"><p> A Role for Snu114p in Regulating Brr2p-dependent Spliceosome Dynamics A simplified splicing cycle… </p></div>
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Figure 6
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<div class="figure-caption-contents">A Role for Snu114p in Regulating Brr2p-dependent Spliceosome Dynamics A simplified splicing cycle is shown, depicting the stages of spliceosome binding and activation (left) and splicing and spliceosome disassembly (right). A model for the roles of Snu114p and Brr2p is illustrated. In the model, Snu114p acts as switch that turns Brr2p “on” and “off”. In the GTP state, Snu114p turns Brr2p “on” to trigger spliceosome activation and disassembly (upper left and lower right, respectively). In the GDP state, Snu114p turns Brr2p “off” to repress inappropriate RNA rearrangements (lower left, upper right). GTP hydrolysis and exchange of GDP for GTP serves to toggle the switch, signaling transitions in the splicing cycle that require up- or down-regulation of Brr2p. See Discussion for details.</div>
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