HnRNPK maintains single strand RNA through controlling double-strand RNA in mammalian cells

Although antisense transcription is a widespread event in the mammalian genome, double-stranded RNA (dsRNA) formation between sense and antisense transcripts is very rare and mechanisms that control dsRNA remain unknown. By characterizing the FGF-2 regulated transcriptome in normal and cancer cells, we identified sense and antisense transcripts IER3 and IER3-AS1 that play a critical role in FGF-2 controlled oncogenic pathways. We show that IER3 and IER3-AS1 regulate each other\u27s transcription through HnRNPK-mediated post-transcriptional regulation. HnRNPK controls the mRNA stability and colocalization of IER3 and IER3-AS1. HnRNPK interaction with IER3 and IER3-AS1 determines their oncogenic functions by maintaining them in a single-stranded form. hnRNPK depletion neutralizes their oncogenic functions through promoting dsRNA formation and cytoplasmic accumulation. Intriguingly, hnRNPK loss-of-function and gain-of-function experiments reveal its role in maintaining global single- and double-stranded RNA. Thus, our data unveil the critical role of HnRNPK in maintaining single-stranded RNAs and their physiological functions by blocking RNA-RNA interactions

Dynein Light Intermediate Chain 2 Facilitates the Metaphase to Anaphase Transition by Inactivating the Spindle Assembly Checkpoint.

The multi-functional molecular motor cytoplasmic dynein performs diverse essential roles during mitosis. The mechanistic importance of the dynein Light Intermediate Chain homologs, LIC1 and LIC2 is unappreciated, especially in the context of mitosis. LIC1 and LIC2 are believed to exist in distinct cytoplasmic dynein complexes as obligate subunits. LIC1 had earlier been reported to be required for metaphase to anaphase progression by inactivating the kinetochore-microtubule attachment-sensing arm of the spindle assembly checkpoint (SAC). However, the functional importance of LIC2 during mitosis remains elusive. Here we report prominent novel roles for the LIC2 subunit of cytoplasmic dynein in regulating the spindle assembly checkpoint. LIC2 depletion in mammalian cells led to prolonged metaphase arrest in the presence of an active SAC and also to stretched kinetochores, thus implicating it in SAC inactivation. Quantitative fluorescence microscopy of SAC components revealed accumulation of both attachment- and tension-sensing checkpoint proteins at metaphase kinetochores upon LIC2 depletion. These observations support a stronger and more diverse role in checkpoint inactivation for LIC2 in comparison to its close homolog LIC1. Our study uncovers a novel functional hierarchy during mitotic checkpoint inactivation between the closely related but homologous LIC subunits of cytoplasmic dynein. These subtle functional distinctions between dynein subpopulations could be exploited to study specific aspects of the spindle assembly checkpoint, which is a key mediator of fidelity in eukaryotic cell division

Bus Guide: An Android Application

ABSTRACT Bus Guide is an android application that tracks a bus at real-time and gather

A model for key LIC2 functions in SAC silencing during mitosis.

<p>Novel functions of mitotic LIC2 uncovered from this study in silencing the spindle assembly checkpoint (SAC) are shown. LIC2-dynein strips attachment sensing SAC proteins (Mad1, Mad2, Zw10) from metaphase kinetochores, like LIC1-dynein. LIC2-dynein has the additional capability of causing removal of tension-sensing SAC protein BubR1, which is lacking in LIC1-dynein. The model offers a possible mechanistic explanation for the differential effects of the two LICs in mediating metaphase to anaphase progression.</p

LIC2 depletion leads to prolonged metaphase arrest.

<p>A) Still images from a movie featuring four different representative siRNA treated Hela cells (control, LIC2 and LIC 1 + 2) showing prolonged arrest in metaphase, followed by delayed anaphase onset (LIC2 > 80 min) or cell death (LIC2 > 4 hrs). B) Average metaphase to anaphase timing, (3 experiments, n = at least 100 cells per experiment). NEB = Nuclear Envelope Breakdown. Statistical significance was calculated by the T-test between two groups. C) Fraction of mitotic cells arrested for more than 80 minutes in metaphase upon respective siRNA treatment. (3 experiments, n = at least 100 cells per experiment). D) Fractions of metaphase cells dying after prolomged metaphase arrest (> 4 hrs) from the cells in C. p values in all panels are * p <0.05, ** p<0.01, *** p<0.001. Scalebar is 75 μm in all images as indicated.</p

LIC2 is required for metaphase to anaphase progression.

<p>A) Metaphase index (% of total cells present in metaphase <b>+/-</b> SD) in Hela cells treated with respective siRNAs as indicated. (3 experiments, n = approximately 500 cells per experiment). B) & C) Western blots showing siRNA mediated LIC1 and LIC2 specific knockdown. LIC2a and LIC2b represent two different siRNA sequences against human LIC2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159646#pone.0159646.ref023" target="_blank">23</a>]. The band marked by the arrow represents human LIC2 that gets reduced upon siRNA treatment, the upper bands are non-specifically recognized by the LIC2 antibody (ThermoScientific). Actin = loading control. D) Rescue of the LIC2-depletion induced metaphase arrest by transgenic expression of rat LIC2 (3 experiments, n = at least 500 cells per experiment). E) Western blots showing specific depletion of LIC2 upon treatment with LIC2-specific siRNAs, using a different LIC2 antibody (Abcam). LIC2a and LIC2b represent two different siRNA sequences against human LIC2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159646#pone.0159646.ref023" target="_blank">23</a>]. The band marked by the arrow represents human LIC2 that appears at the same molecular weight as in C.</p

LIC2 acts on the spindle assembly checkpoint (SAC) to ensure mitotic progression.

<p>A) Metaphase index in Hela cells after respective siRNA treatments of Mad2 and BubR1 alone or co-depleted with LIC2, as indicated on the x-axis (3 experiments, n = at least 200 cells per experiment). *** p<0.001, ns = not significant (p > 0.05). B) Western blot showing depletion of Mad2 by siRNA treatment. Actin = loading control.</p

LIC2 depletion leads to increased inter-kinetochore tension in metaphase.

<p>A) Confocal immunofluorescence images of metaphase Hela cells stained for kinetochores (red, CREST) and chromatin (DAPI). White lines indicate representative distance measurements between sister kinetochores. The insets on the right are zoomed to equivalent levels in both upper and lower panels. B) Average inter-kinetochore distance (μm) on y-axis. 3 experiments; n = at least 30 metaphase cells per experiment (10 pairs of sister chromatids measured per cell). ** = p < 0.01). Scalebar is 5 μm in all images that are not zoomed.</p

Localization of LICs in Mitosis.

<p>A) Confocal immunofluorescence images of Hela cells depicting the localization of LIC1 (red) and LIC2 (green), shown by arrows. B) Fluorescence images of Hela cells showing the presence of LIC2 (green, using the ThermoScientific antibody) at kinetochores (CREST, red) in prometaphase. C) Fluorescence imaging after treatment of cells with anti-LIC2 siRNA, showing loss of LIC2 signal from spindle poles and kinetochores (arrow in inset). DAPI was used to visualize chromosomes. Scalebar is 5 μm in all images that are not zoomed.</p