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1.  The Human Nuclear Poly(A)-Binding Protein Promotes RNA Hyperadenylation and Decay 
PLoS Genetics  2013;9(10):e1003893.
Control of nuclear RNA stability is essential for proper gene expression, but the mechanisms governing RNA degradation in mammalian nuclei are poorly defined. In this study, we uncover a mammalian RNA decay pathway that depends on the nuclear poly(A)-binding protein (PABPN1), the poly(A) polymerases (PAPs), PAPα and PAPγ, and the exosome subunits RRP6 and DIS3. Using a targeted knockdown approach and nuclear RNA reporters, we show that PABPN1 and PAPα, redundantly with PAPγ, generate hyperadenylated decay substrates that are recognized by the exosome and degraded. Poly(A) tail extension appears to be necessary for decay, as cordycepin treatment or point mutations in the PAP-stimulating domain of PABPN1 leads to the accumulation of stable transcripts with shorter poly(A) tails than controls. Mechanistically, these data suggest that PABPN1-dependent promotion of PAP activity can stimulate nuclear RNA decay. Importantly, efficiently exported RNAs are unaffected by this decay pathway, supporting an mRNA quality control function for this pathway. Finally, analyses of both bulk poly(A) tails and specific endogenous transcripts reveals that a subset of nuclear RNAs are hyperadenylated in a PABPN1-dependent fashion, and this hyperadenylation can be either uncoupled or coupled with decay. Our results highlight a complex relationship between PABPN1, PAPα/γ, and nuclear RNA decay, and we suggest that these activities may play broader roles in the regulation of human gene expression.
Author Summary
In eukaryotes, mRNAs include a stretch of adenosine nucleotides at their 3′ end termed the poly(A) tail. In the cytoplasm, the poly(A) tail stimulates translation of the mRNA into protein, and protects the transcript from degradation. Evidence suggests that poly(A) tails may play distinct roles in RNA metabolism in the nucleus, but little is known about these functions and mechanisms. We show here that poly(A) tails can stimulate transcript decay in the nucleus, a function mediated by the ubiquitous nuclear poly(A) binding protein PABPN1. We find that PABPN1 is required for the degradation of a viral nuclear noncoding RNA as well as an inefficiently exported human mRNA. Importantly, the targeting of RNAs to this decay pathway requires the PABPN1 and poly(A) polymerase-dependent extension of the poly(A) tail. Nuclear transcripts with longer poly(A) tails are then selectively degraded by components of the nuclear exosome. These studies elucidate mechanisms that mammalian cells use to ensure proper mRNA “quality control” and may be important to regulate the expression of nuclear noncoding RNAs. Furthermore, our results suggest that the poly(A) tail has diverse and context-specific roles in gene expression.
doi:10.1371/journal.pgen.1003893
PMCID: PMC3798265  PMID: 24146636
2.  The assembly of a GTPase–kinase signalling complex by a bacterial catalytic scaffold 
Nature  2010;469(7328):107-111.
The fidelity and specificity of information flow within a cell is controlled by scaffolding proteins that assemble and link enzymes into signalling circuits1,2. These circuits can be inhibited by bacterial effector proteins that post-translationally modify individual pathway components3–6. However, there is emerging evidence that pathogens directly organize higher-order signalling networks through enzyme scaffolding7,8, and the identity of the effectors and their mechanisms of action are poorly understood. Here we identify the enterohaemorrhagic Escherichia coli O157:H7 type III effector EspG as a regulator of endomembrane trafficking using a functional screen, and report ADP-ribosylation factor (ARF) GTPases and p21-activated kinases (PAKs) as its relevant host substrates. The 2.5 Å crystal structure of EspG in complex with ARF6 shows how EspG blocks GTPase-activating-protein-assisted GTP hydrolysis, revealing a potent mechanism of GTPase signalling inhibition at organelle membranes. In addition, the 2.8 Å crystal structure of EspG in complex with the autoinhibitory Iα3-helix of PAK2 defines a previously unknown catalytic site in EspG and provides an allosteric mechanism of kinase activation by a bacterial effector. Unexpectedly, ARF and PAKs are organized on adjacent surfaces of EspG, indicating its role as a ‘catalytic scaffold’ that effectively reprograms cellular events through the functional assembly of GTPase-kinase signalling complex.
doi:10.1038/nature09593
PMCID: PMC3675890  PMID: 21170023
3.  Defining the pathway of cytoplasmic maturation of the 60S ribosomal subunit 
Molecular cell  2010;39(2):196-208.
In eukaryotic cells the final maturation of ribosomes occurs in the cytoplasm, where trans-acting factors are removed and critical ribosomal proteins are added for functionality. Here, we have carried out a comprehensive analysis of cytoplasmic maturation, ordering the known steps into a coherent pathway. Maturation is initiated by the ATPase Drg1. Downstream, assembly of the ribosome stalk is essential for the release of Tif6. The stalk recruits GTPases during translation. Because the GTPase Efl1, which is required for the release of Tif6, resembles the translation elongation factor eEF2, we suggest that assembly of the stalk recruits Efl1, triggering a step in 60S biogenesis that mimics aspects of translocation. Efl1 could thereby provide a mechanism to functionally check the nascent subunit. Finally, the release of Tif6 is a prerequisite for the release of the nuclear export adapter Nmd3. Establishing this pathway provides an important conceptual framework for understanding ribosome maturation.
doi:10.1016/j.molcel.2010.06.018
PMCID: PMC2925414  PMID: 20670889
ribosome; ribosome biogenesis; EFL1; NMD3; TIF6

Results 1-3 (3)