Research

 

Human RNA machines and their roles in disease: ALS and cancer

 

 

Examples of ongoing projects include the following:

1. The motor neuron diseases ALS and SMA are linked at the molecular level.

ALS is a progressive, adult-onset motor neuron disease that leads to paralysis and is fatal within 2-5 years.  ALS shares clinical features with the motor neuron disease Spinal Muscular Atrophy (SMA), which strikes in childhood.  Mutations in the RNA binding proteins FUS and TDP-43 are causes of ALS whereas a deficiency of the SMN protein causes SMA.  SMN is a component of the SMN complex, which localizes in the cytoplasm as well as in nuclear bodies known as Gems.  Loss of Gems is a hallmark of SMA patient cells.  Strikingly, we found that Gems are deficient in ALS patient cells bearing FUS or TDP-43 mutations.  Together, this work indicates that ALS and SMA are linked via a shared molecular pathway involving TDP-43, FUS, SMN, and Gems (Yamazaki et al., Cell Reports 2012: 2, 799).  We are currently exploring the generality of the Gem phenotype in additional types of ALS and conducting a high throughput screen to identify drug candidates for the disease. 

 

2. The anti-tumor drug E7107* targets U2 snRNP, an essential component of the spliceosome.

The vast majority of human pre-mRNAs contain numerous introns, which must be precisely excised by the spliceosome in order to generate functional mRNAs.  The spliceosome is a multi-component RNA machine, which contains five snRNPs (U1, U2, U4, U5, and U6 snRNPs). The snRNPs bind to the intron in a highly orchestrated series of steps during which critical base-pairing interactions are formed between the snRNAs and the intron.  In one of these interactions, U2 snRNA base pairs to a specific region in the intron known as the branchpoint sequence (BPS). Previous work revealed that U2 snRNP is the target of the anti-tumor drug E7107 (Kotake et al, Nat Chem Biol 2007: 3, 570).  To further define the mechanism of action of E7107, we carried out in vitro studies using our coupled RNAP II transcription/splicing system (Das et al, Genes Dev 2006: 20, 1100, Das et al, Mol Cell 2007: 26, 867, Yu et al, NAR 2010: 38, 7570).  This analysis revealed that E7107 results in assembly of defective spliceosomes in which tight binding of U2 snRNP does not occur (Folco et al, Genes Dev 2011: 25, 440).  Our data indicate that E7107 prevents this tight binding by blocking an ATP-dependent remodeling event that is required to expose the region of U2 snRNA that base pairs to the BPS.  Gaining insight into the mechanism of action of E7107 is an important step towards determining how this drug targets cancer cells.

*The E7107 was provided by Eisai Co., Ltd.

 

3. The TREX complex: A conserved machine for exporting mRNA from the nucleus to the cytoplasm. 

Expression of protein-coding genes is a highly coordinated process beginning with RNAP II transcription in the nucleus to generate pre-mRNA, which then undergoes 5’ end capping, splicing, and 3’ end formation.  The mature mRNA is then exported to the cytoplasm for translation into protein.  We previously identified the TREX complex as the main machinery for packaging mRNA for export (Masuda et al, Genes Dev 2005: 19, 1512, Stasser et al, Nature 2002: 417, 304).  More recently, we characterized the human TREX complex in detail, which led to the identification of the tumorogenic protein, CIP29, as a new component (Dufu et al, Genes Dev 2010: 24, 2043).  In addition, we identified several other new TREX components, including SRAG, POLDIP, and ZC11A (Folco et al, PLoS One 2012: 7, e43804).  Remarkably, all of these proteins interact in an ATP-dependent manner with UAP56, which is the only component of TREX that is an ATP-dependent RNA helicase.  Together, these studies indicate that the TREX complex is dynamically remodeled in an ATP-dependent manner, and further work is essential to understand how this remodeling plays a role in packaging and exporting mRNA.

 

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