Nimisha Sharma

Mechanism of Ca²⁺-triggered ESCRT assembly and regulation of cell membrane repair.

In muscle and other mechanically active tissue, cell membranes are constantly injured and their repair depends on the injury induced increase in cytosolic calcium. Here we show that injury-triggered Ca2+ increase results in assembly of ESCRTIII and accessory proteins at the site of repair. This process is initiated by the calcium binding protein - Apoptosis Linked Gene (ALG)-2. ALG-2 facilitates accumulation of ALG-2 interacting protein X (ALIX), ESCRT III, and Vps4 complex at the injured cell membrane, which in turn results in cleavage and shedding of the damaged part of the cell membrane. Lack of ALG-2, ALIX, or Vps4B each prevents shedding, and repair of the injured cell membrane. These results demonstrate Ca2+-dependent accumulation of ESCRTIII-Vps4 complex following large focal injury to the cell membrane and identify the role of ALG-2 as the initiator of sequential ESCRTIII-Vps4 complex assembly that facilitates scission and repair of the injured cell membrane.

Use of quantitative membrane proteomics identifies a novel role of mitochondria in healing injured muscles.

Background: Cellular processes involved in healing injured skeletal muscle fibers are poorly understood. Results: Using an improved quantitative membrane proteomics approach for cells and tissues, we have identified accumulation of mitochondria at the site of sarcolemma injury as a key requirement for myofiber healing. Conclusion: Mitochondria are the earliest responders to myofiber injury. Significance: This work identifies a novel function of mitochondria in muscle injury. Skeletal muscles are proficient at healing from a variety of injuries. Healing occurs in two phases, early and late phase. Early phase involves healing the injured sarcolemma and restricting the spread of damage to the injured myofiber. Late phase of healing occurs a few days postinjury and involves interaction of injured myofibers with regenerative and inflammatory cells. Of the two phases, cellular and molecular processes involved in the early phase of healing are poorly understood. We have implemented an improved sarcolemmal proteomics approach together with in vivo labeling of proteins with modified amino acids in mice to study acute changes in the sarcolemmal proteome in early phase of myofiber injury. We find that a notable early phase response to muscle injury is an increased association of mitochondria with the injured sarcolemma. Real-time imaging of live myofibers during injury demonstrated that the increased association of mitochondria with the injured sarcolemma involves translocation of mitochondria to the site of injury, a response that is lacking in cultured myoblasts. Inhibiting mitochondrial function at the time of injury inhibited healing of the injured myofibers. This identifies a novel role of mitochondria in the early phase of healing injured myofibers.

The fission yeast Rpb4 subunit of RNA polymerase II plays a specialized role in cell separation

RNA polymerase II is a complex of 12 subunits, Rpb1 to Rpb12, whose specific roles are only partly understood. Rpb4 is essential in mammals and fission yeast, but not in budding yeast. To learn more about the roles of Rpb4, we expressed the rpb4 gene under the control of regulatable promoters of different strength in fission yeast. We demonstrate that below a critical level of transcription, Rpb4 affects cellular growth proportional to its expression levels: cells expressing lower levels of rpb4 grew slower compared to cells expressing higher levels. Lowered rpb4 expression did not affect cell survival under several stress conditions, but it caused specific defects in cell separation similar to sep mutants. Microarray analysis revealed that lowered rpb4 expression causes a global reduction in gene expression, but the transcript levels of a distinct subset of genes were particularly responsive to changes in rpb4 expression. These genes show some overlap with those regulated by the Sep1-Ace2 transcriptional cascade required for cell separation. Most notably, the gene expression signature of cells with lowered rpb4 expression was highly similar to those of mcs6, pmh1, sep10 and sep15 mutants. Mcs6 and Pmh1 encode orthologs of metazoan TFIIH-associated cyclin-dependent kinase (CDK)-activating kinase (Cdk7-cyclin H-Mat1), while Sep10 and Sep15 encode mediator components. Our results suggest that Rpb4, along with some other general transcription factors, plays a specialized role in a transcriptional pathway that controls the cell cycle-regulated transcription of a specific subset of genes involved in cell division.

The Med8 mediator subunit interacts with the Rpb4 subunit of RNA polymerase II and Ace2 transcriptional activator in Schizosaccharomyces pombe

MINT‐7260735: rpb4 (uniprotkb:O74825) binds (MI:0407) to med8 (uniprotkb:O94646) by pull down (MI:0096)

Overexpression of the gene for Rpb7 subunit of yeast RNA polymerase II rescues the phenotypes associated with absence of the largest, nonessential subunit Rpb4

The easily dissociable subcomplex of Rpb4 and Rpb7 subunits of yeast RNA polymerase II has been considered, for long, to play a role in stabilizing Pol II under stress. On the basis of previous genetic and biochemical observations, it was proposed that within the subcomplex one of the functions of Rpb4p is to stabilize the interaction between Rpb7p and the rest of Pol II. We took a direct approach to test the latter possibility by overexpression and mutagenesis ofRPB7 in absence of Rpb4p. We report here the results, which support the latter hypothesis. While it has been previously reported that absence of Rpb4p results in reduction in overall transcription by Pol II, our comparative analysis of RNAs fromRPB4 andrpb4δ cells suggests that there are indeed several genes differentially expressed between the two cells. We propose that the qualitative differences in overall transcription in presence and absence of Rpb4p imply a more active role for Rpb4p in transcription of at least a subset of genes.

Rpb4 and Rpb7: multifunctional subunits of RNA polymerase II

The 12-subunit RNA polymerase II enzyme in yeasts and higher eukaryotic cells is important for transcription of protein-coding genes. Its fourth and seventh largest subunits named Rpb4 and Rpb7, respectively, display some unique features that distinguish them from the remaining subunits of this enzyme. These two subunits also bind to each other forming a complex in archaebacteria, yeasts, plants and humans. Our knowledge about the structure and functions of this complex has greatly advanced in recent years. These subunits were initially considered to be important only for initiation of transcription and stress response. However, recent evidence suggests that they are not only involved in transcription, but also in DNA repair, mRNA export and decay as well as translation, highlighting the roles of this heterodimer in diverse biological processes. In this article, we review the current status of these two subunits and discuss attributes of their structure and function across organisms.

Regulation of RNA polymerase II-mediated transcriptional elongation: Implications in human disease

Expression of protein‐coding genes is primarily regulated at the level of transcription. Most of the earlier studies focussed on understanding the assembly of the pre‐initiation complex at the promoter of genes and subsequent initiation of transcription as the regulatory steps in transcription. However, research over the last decade has demonstrated the significance of regulating transcription of genes at the elongation stage. Several new proteins have been identified that control this step and our knowledge about their functions is expanding rapidly. Moreover, an increasing body of evidence suggests that a dysfunction of these transcription elongation factors is related to several diseases. Here, we review the latest advances in our understanding about the in vivo roles of the transcription elongation factors and their link with diseases.

Modulation of polymerase II composition: A possible mode of transcriptional regulation of stress response in eukaryotes

Regulation of stress response in prokaryotes is mainly achieved at the transcriptional initiation level. Prokaryotes use alternative holoenzymes, consisting of the core polymerase associated with different sigma factors, which confer on it altered specificity of transcriptional initiation. Stress response being probably one of the most inevitable features of life, it would be interesting to find if eukaryotes also use a similar strategy at this level of regulation. Since the yeastSaccharomyces cerevisiae is a model system for studying many different phenomena in eukaryotes we review the transcriptional regulation of stress in this system. Based on published observations in the literature and our own studies, we have analysed the regulation of stress response, in the yeastS. cerevisiae. Two of the core subunits of the yeast RNA polymerase II, which show altered stoichiometry within the polymerase under different conditions appear to be involved specifically in regulating the stress response. In a very broad sense then, the altered subunit composition of the core polymerase or a different holoenzyme, appears to correlate with gene expression specific to stress response inS. cerevisiae and probably reflects the scenario in other eukaryotes.

Schizosaccharomyces pombe Pol II transcription elongation factor ELL functions as part of a rudimentary super elongation complex

Abstract ELL family transcription factors activate the overall rate of RNA polymerase II (Pol II) transcription elongation by binding directly to Pol II and suppressing its tendency to pause. In metazoa, ELL regulates Pol II transcription elongation as part of a large multisubunit complex referred to as the Super Elongation Complex (SEC), which includes P-TEFb and EAF, AF9 or ENL, and an AFF family protein. Although orthologs of ELL and EAF have been identified in lower eukaryotes including Schizosaccharomyces pombe, it has been unclear whether SEC-like complexes function in lower eukaryotes. In this report, we describe isolation from S. pombe of an ELL-containing complex with features of a rudimentary SEC. This complex includes S. pombe Ell1, Eaf1, and a previously uncharacterized protein we designate Ell1 binding protein 1 (Ebp1), which is distantly related to metazoan AFF family members. Like the metazoan SEC, this S. pombe ELL complex appears to function broadly in Pol II transcription. Interestingly, it appears to have a particularly important role in regulating genes involved in cell separation.

Structure function characterization of the ELL Associated Factor (EAF) from Schizosaccharomyces pombe

EAF (ELL Associated Factor) proteins interact with the transcription elongation factor, ELL (Eleven nineteen Lysine rich Leukemia) and enhance its ability to stimulate RNA polymerase II-mediated transcriptional elongation in vitro. Schizosaccharomyces pombe contains a single homolog of EAF (SpEAF), which is not essential for survival of S. pombe in contrast to its essential higher eukaryotic homologs. The physiological role of SpEAF is not well understood. In this study, we show that S. pombe EAF is important in regulating growth of S. pombe cells during normal growth conditions. Moreover, SpEAF is also essential for survival under conditions of DNA damage, while its deletion does not affect growth under environmental stress conditions. Our in vivo structure-function studies further demonstrate that while both the amino and carboxyl terminal domains of SpEAF possess the potential to activate transcription, only the amino terminal domain of SpEAF is involved in interaction with the S. pombe ELL protein. The carboxyl-terminus of SpEAF is required for rescue of the growth defect under normal and DNA damaging conditions that is associated with the absence of SpEAF. Using bioinformatics and circular dichroism spectroscopy, we show that the carboxyl-terminus of SpEAF has a disordered conformation. Furthermore, addition of trifluoroethanol triggered its transition from a disordered to α-helical conformation. Taken together, the results presented here identify novel structural and functional features of SpEAF protein, providing insights into how EAF proteins may enforce transcriptional control of gene expression.