A Quantitative, High-Throughput Reverse Genetic Screen Reveals Novel Connections between Pre–mRNA Splicing and 5′ and 3′ End Transcript Determinants

Here we present the development and implementation of a genome-wide reverse genetic screen in the budding yeast, Saccharomyces cerevisiae, that couples high-throughput strain growth, robotic RNA isolation and cDNA synthesis, and quantitative PCR to allow for a robust determination of the level of nearly any cellular RNA in the background of 5,500 different mutants. As an initial test of this approach, we sought to identify the full complement of factors that impact pre–mRNA splicing. Increasing lines of evidence suggest a relationship between pre–mRNA splicing and other cellular pathways including chromatin remodeling, transcription, and 3′ end processing, yet in many cases the specific proteins responsible for functionally connecting these pathways remain unclear. Moreover, it is unclear whether all pathways that are coupled to splicing have been identified. As expected, our approach sensitively detects pre–mRNA accumulation in the vast majority of strains containing mutations in known splicing factors. Remarkably, however, several additional candidates were found to cause increases in pre–mRNA levels similar to that seen for canonical splicing mutants, none of which had previously been implicated in the splicing pathway. Instead, several of these factors have been previously implicated to play roles in chromatin remodeling, 3′ end processing, and other novel categories. Further analysis of these factors using splicing-sensitive microarrays confirms that deletion of Bdf1, a factor that links transcription initiation and chromatin remodeling, leads to a global splicing defect, providing evidence for a novel connection between pre–mRNA splicing and this component of the SWR1 complex. By contrast, mutations in 3′ end processing factors such as Cft2 and Yth1 also result in pre–mRNA splicing defects, although only for a subset of transcripts, suggesting that spliceosome assembly in S. cerevisiae may more closely resemble mammalian models of exon-definition. More broadly, our work demonstrates the capacity of this approach to identify novel regulators of various cellular RNAs

Microtubules are necessary for proper Reticulon localization during mitosis.

During mitosis, the structure of the Endoplasmic Reticulum (ER) displays a dramatic reorganization and remodeling, however, the mechanism driving these changes is poorly understood. Hairpin-containing ER transmembrane proteins that stabilize ER tubules have been identified as possible factors to promote these drastic changes in ER morphology. Recently, the Reticulon and REEP family of ER shaping proteins have been shown to heavily influence ER morphology by driving the formation of ER tubules, which are known for their close proximity with microtubules. Here, we examine the role of microtubules and other cytoskeletal factors in the dynamics of a Drosophila Reticulon, Reticulon-like 1 (Rtnl1), localization to spindle poles during mitosis in the early embryo. At prometaphase, Rtnl1 is enriched to spindle poles just prior to the ER retention motif KDEL, suggesting a possible recruitment role for Rtnl1 in the bulk localization of ER to spindle poles. Using image analysis-based methods and precise temporal injections of cytoskeletal inhibitors in the early syncytial Drosophila embryo, we show that microtubules are necessary for proper Rtnl1 localization to spindles during mitosis. Lastly, we show that astral microtubules, not microfilaments, are necessary for proper Rtnl1 localization to spindle poles, and is largely independent of the minus-end directed motor protein dynein. This work highlights the role of the microtubule cytoskeleton in Rtnl1 localization to spindles during mitosis and sheds light on a pathway towards inheritance of this major organelle

Microtubules are necessary for proper Reticulon localization during mitosis.

During mitosis, the structure of the Endoplasmic Reticulum (ER) displays a dramatic reorganization and remodeling, however, the mechanism driving these changes is poorly understood. Hairpin-containing ER transmembrane proteins that stabilize ER tubules have been identified as possible factors to promote these drastic changes in ER morphology. Recently, the Reticulon and REEP family of ER shaping proteins have been shown to heavily influence ER morphology by driving the formation of ER tubules, which are known for their close proximity with microtubules. Here, we examine the role of microtubules and other cytoskeletal factors in the dynamics of a Drosophila Reticulon, Reticulon-like 1 (Rtnl1), localization to spindle poles during mitosis in the early embryo. At prometaphase, Rtnl1 is enriched to spindle poles just prior to the ER retention motif KDEL, suggesting a possible recruitment role for Rtnl1 in the bulk localization of ER to spindle poles. Using image analysis-based methods and precise temporal injections of cytoskeletal inhibitors in the early syncytial Drosophila embryo, we show that microtubules are necessary for proper Rtnl1 localization to spindles during mitosis. Lastly, we show that astral microtubules, not microfilaments, are necessary for proper Rtnl1 localization to spindle poles, and is largely independent of the minus-end directed motor protein dynein. This work highlights the role of the microtubule cytoskeleton in Rtnl1 localization to spindles during mitosis and sheds light on a pathway towards inheritance of this major organelle

Arresting the <i>Drosophila</i> embryo in interphase maintains the ER in an interphase-like state.

<p>(<b>A</b>) Time-lapse confocal images of a Pdi-GFP (green) / H2-RFP (red) transgenic embryo injected at metaphase cycle 10 with the DNA replication inhibitor, aphidicolin (APH) and viewed during cycle 11. APH, arrests the embryo in S-phase of cycle 11. In the presence of APH, the ER displayed a loose uniform distribution around the nuclei denoting an interphase-like state. This interphase-like state of the ER persists for greater than 30 minutes without any changes to either localization or structure. This is quantified in the fluorescence intensity traces below (see yellow dotted-lines in merged images). H2-RFP signal inside the nucleus does not increase over this time period as well (arrowheads). (<b>B</b>) Time-lapse confocal images of a Pdi-GFP (green) / H2-RFP (red) embryo injected with the protein synthesis inhibitor cycloheximide (CHX) at metaphase of cycle 10 and viewed during interphase of the following cycle. Similar to APH, CHX induced arrest which maintained the ER in an interphase-like state. This is quantified below, as in A. (<b>C</b>) Similar background and approach as A and B. Embryos were injected with an APH+CHX cocktail. ER membrane maintained an interphase-like organization as seen in APH injections alone. Scale bar is 10 μm. Time is in min:sec.</p

3D reconstruction of ER structural changes display a clustering of extended cisternae at the spindle poles during metaphase.

<p>Embryos expressing Pdi-GFP (green) and H2-RFP (red) were fixed and imaged using confocal microscopy. (<b>A</b>) Upper panels (view 1) represent a top view of the nucleus and surrounding ER along the xy-plane and bottom panels (view 2) show the nucleus and ER ~45° -75° tilt along the in the y-plane. Embryos were imaged in the z-direction with a step size of 0.1 μm and subject to 3D reconstruction software. (<b>B</b>) At telophase of cycle 11, the ER is globular and spread along the reforming nuclear envelope and at the midbody (view 1, arrowhead). Exiting mitosis, at interphase, the ER is spread loosely through the cytoplasm outline the nuclear envelope. At prophase, the ER becomes defined and begins to cluster and propagate apically at the spindle poles. These clusters are not uniform in size and appear to be sheet-like structures (view 2, arrows). At metaphase, the clusters are found at the spindle poles and appear to be connected along the spindle area forming a sheath (view 1, arrow). In anaphase, ER cisternal clusters appear with the segregating chromosomes and at the midbody (view 2, arrow). Scale bar is 5 μm.</p

The ER displays dramatic structural and localization changes during mitosis in the early <i>Drosophila</i> embryo.

<p>(<b>A</b>) Mitotic ER dynamics were examined in cycle 11 transgenic <i>Drosophila</i> embryos expressing the ER marker Pdi-GFP and the DNA marker H2-RFP using time-lapse confocal microscopy. Phases of mitosis are listed at the top with relative time (min:sec) listed in the merge panels. During interphase, ER (green) was initially spread loosely around the nucleus. Upon entry into mitosis, ER accumulates around the nucleus and was rapidly converted to thick, perinuclear cisternae upon chromosome condensation (red) and prophase onset. in prometaphase, the ER membrane reorganizes with the developing mitotic spindle and begins to accumulate at the spindle poles. At metaphase and anaphase the ER is aligned with the mitotic spindle and displays movement towards the spindle poles (arrow). During late anaphase and telophase, the ER sees a rapid localization around the segregated, decondensing chromosomes and a localization at the central spindle / midbody (arrowhead). Scale bar is 10 μm. (<b>B</b>) High magnification of mitotic ER changes following a single nucleus used for quantification of ER movements shown in C (asterisk in A). Yellow line denotes fluorescence trace shown in C. Scale bar is 5 μm. (<b>C</b>) Fluorescence intensity trace of ER (green line) and chromosomes (red line) along 20 μm of the developing embryo. ER fluorescence is maximal just adjacent to the nuclear space, but is excluded from the nucleus. During interphase, the ER is evenly distributed throughout the cytoplasm. Intensity around the nucleus increases during mitosis and follows the extension of the spindle. Pdi-GFP signal intensity reached maximum during metaphase (arrows). Condensation and alignment of chromosomes at the metaphase plate are marked by the arrowhead. At telophase, two new nuclear envelopes are formed with a large peak at the remaining midbody.</p

Inhibition of the APC/C maintains the ER in a mitotic state.

<p>(<b>A</b>) Time-lapse confocal images of a cycle 10 Pdi-GFP / H2-RFP transgenic embryo following micro-injection of a dominant-negative form of UbcH10 just prior to entry into mitosis, eventually arresting the embryo in metaphase. The ER displayed normal structural organization and localization changes early in mitosis and relocated to the mitotic spindle upon nuclear envelope breakdown. The embryo then arrested at metaphase and the ER remained along the mitotic spindle. Flares of membrane began to protrude from the perispindle area. ER membrane was steadily lost from the area adjacent to the spindle pole over time (~20 minutes). Yellow trace line indicates plots for B, orange line for C. Scale bar is 5 μm. Time is in min:sec. (<b>B</b>) Fluorescence intensity trace plots of the longitudinal section of a nucleus. The plots are similar to wildtype through metaphase (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117859#pone.0117859.g001" target="_blank">Fig. 1C</a>). The later time points show a lack of intense ER at the poles, as seen in wildtype. (<b>C</b>) A plot of fluorescence intensity through a latitudinal section of the spindle highlights the increase in fluorescence around the spindle normally seen during mitosis. At the arrest, multiple peaks are seen where flares of ER expand from the perispindle region (arrows).</p

Mitotic ER rearrangements do not occur until after NEB.

<p>(<b>A</b>) An embryo expressing Rtnl1-GFP and mCh-Tub was observed during cycle 11. Tubulin entered the nuclear space at the 20 second time-point, signaling nuclear envelope breakdown prior to accumulation of ER at the centrosome. At the 40 second time point ER began its rearrangement at the centrosome (arrow). Yellow dotted line denotes fluorescence trace shown in B. Scale bar is 5 μm. (<b>B</b>) Fluorescence intensity traces of Rtnl1 (green line) and mCh-Tub (red line). mCh-Tub displays an intensity peak at 5 μm and 10 μm before NEB (arrowheads). At 20 seconds, mCh-Tub intensity becomes flat indicating NEB, while Rtnl1 intensity begins to form peaks at 5 μm and 10 μm (arrows). Rtnl1 intensity continues to rise and mCh-Tub intensity also rises between 5 μm and 10 μm indicating mitotic spindle formation. Time is in min:sec.</p