Maternal SIN3A regulates reprogramming of gene expression during mouse preimplantation development.

The oocyte-to-embryo transition entails genome activation and a dramatic reprogramming of gene expression that is required for continued development. Superimposed on genome activation and reprogramming is development of a transcriptionally repressive state at the level of chromatin structure. Inducing global histone hyperacetylation relieves this repression and histone deacetylases 1 and 2 (HDAC1 and HDAC2) are involved in establishing the repressive state. Because SIN3A is an HDAC1/2-containing complex, we investigated whether it is involved in reprogramming gene expression during the course of genome activation. We find that Sin3a mRNA is recruited during maturation and that inhibiting its recruitment not only inhibits development beyond the 2-cell stage but also compromises the fidelity of reprogramming gene expression. The SIN3A that is synthesized during oocyte maturation reaches a maximum level in the mid-1-cell embryo and is essentially absent by the mid-2-cell stage. Overexpressing SIN3A in 1-cell embryos has no obvious effect on pre- and postimplantation development. These results provide a mechanism by which reprogramming can occur using a maternally inherited transcription machinery, namely, recruitment of mRNAs encoding transcription factors and chromatin remodelers, such as SIN3A.

Essential Role for endogenous siRNAs during meiosis in mouse oocytes.

The RNase III enzyme DICER generates both microRNAs (miRNAs) and endogenous short interfering RNAs (endo-siRNAs). Both small RNA species silence gene expression post-transcriptionally in association with the ARGONAUTE (AGO) family of proteins. In mammals, there are four AGO proteins (AGO1-4), of which only AGO2 possesses endonucleolytic activity. siRNAs trigger endonucleolytic cleavage of target mRNAs, mediated by AGO2, whereas miRNAs cause translational repression and mRNA decay through association with any of the four AGO proteins. Dicer deletion in mouse oocytes leads to female infertility due to defects during meiosis I. Because mouse oocytes express both miRNAs and endo-siRNAs, this phenotype could be due to the absence of either class of small RNA, or both. However, we and others demonstrated that miRNA function is suppressed in mouse oocytes, which suggested that endo-siRNAs, not miRNAs, are essential for female meiosis. To determine if this was the case we generated mice that express a catalytically inactive knock-in allele of Ago2 (Ago2ADH) exclusively in oocytes and thereby disrupted the function of siRNAs. Oogenesis and hormonal response are normal in Ago2ADH oocytes, but meiotic maturation is impaired, with severe defects in spindle formation and chromosome alignment that lead to meiotic catastrophe. The transcriptome of these oocytes is widely perturbed and shows a highly significant correlation with the transcriptome of Dicer null and Ago2 null oocytes. Expression of the mouse transcript (MT), the most abundant transposable element in mouse oocytes, is increased. This study reveals that endo-siRNAs are essential during meiosis I in mouse females, demonstrating a role for endo-siRNAs in mammals.

Increased CDK1 activity determines the timing of kinetochore-microtubule attachments in meiosis I.

Chromosome segregation during cell division depends on stable attachment of kinetochores to spindle microtubules. Mitotic spindle formation and kinetochore-microtubule (K-MT) capture typically occur within minutes of nuclear envelope breakdown. In contrast, during meiosis I in mouse oocytes, formation of the acentrosomal bipolar spindle takes 3-4 h, and stabilization of K-MT attachments is delayed an additional 3-4 h. The mechanism responsible for this delay, which likely prevents stabilization of erroneous attachments during spindle formation, is unknown. Here we show that during meiosis I, attachments are regulated by CDK1 activity, which gradually increases through prometaphase and metaphase I. Partial reduction of CDK1 activity delayed formation of stable attachments, whereas a premature increase in CDK1 activity led to precocious formation of stable attachments and eventually lagging chromosomes at anaphase I. These results indicate that the slow increase in CDK1 activity in meiosis I acts as a timing mechanism to allow stable K-MT attachments only after bipolar spindle formation, thus preventing attachment errors.

Polo-like kinase-1 regulates kinetochore-microtubule dynamics and spindle checkpoint silencing.

Polo-like kinase-1 (Plk1) is a highly conserved kinase with multiple mitotic functions. Plk1 localizes to prometaphase kinetochores and is reduced at metaphase kinetochores, similar to many checkpoint signaling proteins, but Plk1 is not required for spindle checkpoint function. Plk1 is also implicated in stabilizing kinetochore-microtubule attachments, but these attachments are most stable when kinetochore Plk1 levels are low at metaphase. Therefore, it is unclear how Plk1 function at kinetochores can be understood in the context of its dynamic localization. In this paper, we show that Plk1 activity suppresses kinetochore-microtubule dynamics to stabilize initial attachments in prometaphase, and Plk1 removal from kinetochores is necessary to maintain dynamic microtubules in metaphase. Constitutively targeting Plk1 to kinetochores maintained high activity at metaphase, leading to reduced interkinetochore tension and intrakinetochore stretch, a checkpoint-dependent mitotic arrest, and accumulation of microtubule attachment errors. Together, our data show that Plk1 dynamics at kinetochores control two critical mitotic processes: initially establishing correct kinetochore-microtubule attachments and subsequently silencing the spindle checkpoint.

Maternally recruited Aurora C kinase is more stable than Aurora B to support mouse oocyte maturation and early development.

Aurora kinases are highly conserved, essential regulators of cell division. Two Aurora kinase isoforms, A and B (AURKA and AURKB), are expressed ubiquitously in mammals, whereas a third isoform, Aurora C (AURKC), is largely restricted to germ cells. Because AURKC is very similar to AURKB, based on sequence and functional analyses, why germ cells express AURKC is unclear. We report that Aurkc(-/-) females are subfertile, and that AURKB function declines as development progresses based on increasing severity of cytokinesis failure and arrested embryonic development. Furthermore, we find that neither Aurkb nor Aurkc is expressed after the one-cell stage, and that AURKC is more stable during maturation than AURKB using fluorescently tagged reporter proteins. In addition, Aurkc mRNA is recruited during maturation. Because maturation occurs in the absence of transcription, posttranscriptional regulation of Aurkc mRNA, coupled with the greater stability of AURKC protein, provides a means to ensure sufficient Aurora kinase activity, despite loss of AURKB, to support both meiotic and early embryonic cell divisions. These findings suggest a model for the presence of AURKC in oocytes: that AURKC compensates for loss of AURKB through differences in both message recruitment and protein stability.

Emerging roles for the FSH receptor adapter protein APPL1 and overlap of a putative 14-3-3τ interaction domain with a canonical G-protein interaction site.

The interaction of cytoplasmic proteins with intracellular domains of membrane receptors can occur at several opportunities, including: during biosynthesis, while in membrane residency and during internalization and recycling following ligand binding. Since the initial discovery that it interacts with the FSH receptor (FSHR) together with additional members of a potential signaling complex, APPL1 has been shown to interact with a variety of membrane receptors. Recent subcellular localizations of APPL1 place it in dynamic and varied venues in the cell, including at the cell membrane, the nucleus and the early endosomes. Another adapter protein family the 14-3-3 proteins, are largely recognized as binding to phosphorylation sites but recent work demonstrated that in the case of FSHR, the 14-3-3 site overlaps with the canonical G-protein binding site. G-proteins appear to sample the environment and exchange between the membrane and intracellular locales and this binding could be mediated by or modulated by receptor interactions at the 14-3-3 binding site. Observations that multiple proteins can interact with cytoplasmic domains of GPCRs leads to the inescapable conclusion that either the interactions occur via orderly replacement or exchange, or that receptors are simultaneously occupied by a variety of adapters and effectors or even that oligomers of dimeric GPCRs provide for platforms that can simultaneously interact with effectors and adaptors.

Ratiometric bimolecular beacons for the sensitive detection of RNA in single living cells.

Numerous studies have utilized molecular beacons (MBs) to image RNA expression in living cells; however, there is growing evidence that the sensitivity of RNA detection is significantly hampered by their propensity to emit false-positive signals. To overcome these limitations, we have developed a new RNA imaging probe called ratiometric bimolecular beacon (RBMB), which combines functional elements of both conventional MBs and siRNA. Analogous to MBs, RBMBs elicit a fluorescent reporter signal upon hybridization to complementary RNA. In addition, an siRNA-like double-stranded domain is used to facilitate nuclear export. Accordingly, live-cell fluorescent imaging showed that RBMBs are localized predominantly in the cytoplasm, whereas MBs are sequestered into the nucleus. The retention of RBMBs within the cytoplasmic compartment led to >15-fold reduction in false-positive signals and a significantly higher signal-to-background compared with MBs. The RBMBs were also designed to possess an optically distinct reference fluorophore that remains unquenched regardless of probe confirmation. This reference dye not only provided a means to track RBMB localization, but also allowed single cell measurements of RBMB fluorescence to be corrected for variations in probe delivery. Combined, these attributes enabled RBMBs to exhibit an improved sensitivity for RNA detection in living cells.