Sue Lin-Chao

Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays

Much of the information available about factors that affect mRNA decay in Escherichia coli, and by inference in other bacteria, has been gleaned from study of less than 25 of the ≈4,300 predicted E. coli messages. To investigate these factors more broadly, we examined the half-lives and steady-state abundance of known and predicted E. coli mRNAs at single-gene resolution by using two-color fluorescent DNA microarrays. An rRNA-based strategy for normalization of microarray data was developed to permit quantitation of mRNA decay after transcriptional arrest by rifampicin. We found that globally, mRNA half-lives were similar in nutrient-rich media and defined media in which the generation time was approximately tripled. A wide range of stabilities was observed for individual mRNAs of E. coli, although ≈80% of all mRNAs had half-lives between 3 and 8 min. Genes having biologically related metabolic functions were commonly observed to have similar stabilities. Whereas the half-lives of a limited number of mRNAs correlated positively with their abundance, we found that overall, increased mRNA stability is not predictive of increased abundance. Neither the density of putative sites of cleavage by RNase E, which is believed to initiate mRNA decay in E. coli, nor the free energy of folding of 5′ or 3′ untranslated region sequences was predictive of mRNA half-life. Our results identify previously unsuspected features of mRNA decay at a global level and also indicate that generalizations about decay derived from the study of individual gene transcripts may have limited applicability.

High copy number of the pUC plasmid results from a Rom/Rop‐suppressible point mutation in RNA II

The plasmids pUC18 and pUC19 are pBB322 derivatives that replicate at a copy number several fold higher than the parent during growth of Escherichia coli at 37°C. We show here that the high copy number of pUC plasmids results from a single point mutation in the replication primer, RNA II, and that the phenotypic effects of this mutation can be suppressed by the Rom (RNA one modulator)/Rop protein or by lowering the growth temperature to 30°C. The mutations effects are enhanced by cell growth at 42°C, at which copy number is further increased. During normal cell growth, the pUC mutation does not affect the length or function of RNA I, the antisense repressor of plasmid DNA replication, but may, as computer analysis suggests, alter the secondary structure of pUC RNA II. We suggest that the pUC mutation impedes interactions between the repressor and the primer by producing a temperature‐dependent alteration of the RNA II conformation. The Rom/Rop protein may either promote normal folding of the mutated RNA II or, alternatively, may enable the interaction of sub‐optimally folded RNA II with the repressor.

Regulation of ribonuclease E activity by the L4 ribosomal protein of Escherichia coli

Whereas ribosomal proteins (r-proteins) are known primarily as components of the translational machinery, certain of these r-proteins have been found to also have extraribosomal functions. Here we report the novel ability of an r-protein, L4, to regulate RNA degradation in Escherichia coli. We show by affinity purification, immunoprecipitation analysis, and E. coli two-hybrid screening that L4 interacts with a site outside of the catalytic domain of RNase E to regulate the endoribonucleolytic functions of the enzyme, thus inhibiting RNase E-specific cleavage in vitro, stabilizing mRNAs targeted by RNase E in vivo, and controlling plasmid DNA replication by stabilizing an antisense regulatory RNA normally attacked by RNase E. Broader effects of the L4-RNase E interaction on E. coli transcripts were shown by DNA microarray analysis, which revealed changes in the abundance of 65 mRNAs encoding the stress response proteins HslO, Lon, CstA, YjiY, and YaeL, as well as proteins involved in carbohydrate and amino acid metabolism and transport, transcription/translation, and DNA/RNA synthesis. Analysis of mRNA stability showed that the half lives of stress-responsive transcripts were increased by ectopic expression of L4, which normally increases along with other r-proteins in E. coli under stress conditions, and also by inactivation of RNase E. Our finding that L4 can inhibit RNase E-dependent decay may account at least in part for the elevated production of stress-induced proteins during bacterial adaptation to adverse environments.

Stathmin, a microtubule-destabilizing protein, is dysregulated in spinal muscular atrophy

Spinal muscular atrophy (SMA), a motor neuron degeneration disorder, is caused by either mutations or deletions of survival motor neuron 1 (SMN1) gene which result in insufficient SMN protein. Here, we describe a potential link between stathmin and microtubule defects in SMA. Stathmin was identified by screening Smn-knockdown NSC34 cells through proteomics analysis. We found that stathmin was aberrantly upregulated in vitro and in vivo, leading to a decreased level of polymerized tubulin, which was correlated with disease severity. Reduced microtubule densities and beta(III)-tubulin levels in distal axons of affected SMA-like mice and an impaired microtubule network in Smn-deficient cells were observed, suggesting an involvement of stathmin in those microtubule defects. Furthermore, knockdown of stathmin restored the microtubule network defects of Smn-deficient cells, promoted axon outgrowth and reduced the defect in mitochondria transport in SMA-like motor neurons. We conclude that aberrant stathmin levels may play a detrimental role in SMA; this finding suggests a novel approach to treating SMA by enhancing microtubule stability.

Composition and conservation of the mRNA-degrading machinery in bacteria

RNA synthesis and decay counteract each other and therefore inversely regulate gene expression in pro- and eukaryotic cells by controlling the steady-state level of individual transcripts. Genetic and biochemical data together with recent in depth annotation of bacterial genomes indicate that many components of the bacterial RNA decay machinery are evolutionarily conserved and that their functional analogues exist in organisms belonging to all kingdoms of life. Here we briefly review biological functions of essential enzymes, their evolutionary conservation and multienzyme complexes that are involved in mRNA decay in Escherichia coli and discuss their conservation in evolutionarily distant bacteria.

Gas7 Functions with N-WASP to Regulate the Neurite Outgrowth of Hippocampal Neurons

Neuritogenesis, or neurite outgrowth, is a critical process for neuronal differentiation and maturation in which growth cones are formed from highly dynamic actin structures. Gas7 (growth arrest-specific gene 7), a new member of the PCH (Pombe Cdc15 homology) protein family, is predominantly expressed in neurons and is required for the maturation of primary cultured Purkinje neurons as well as the neuron-like differentiation of PC12 cells upon nerve growth factor stimulation. We report that Gas7 co-localizes and physically interacts with N-WASP, a key regulator of Arp2/3 complex-mediated actin polymerization, in the cortical region of Gas7-transfected Neuro-2a cells and growth cones of hippocampal neurons. The interaction between Gas7 and N-WASP is mediated by WW-Pro domains, which is unique in the PCH protein family, where most interactions are of the SH3-Pro kind. The interaction contributes to the formation of membrane protrusions and processes by recruiting the Arp2/3 complex in a Cdc42-independent manner. Importantly, specific interaction between Gas7 and N-WASP is required for regular neurite outgrowth of hippocampal neurons. The data demonstrate an essential role of Gas7 through its interaction with N-WASP during neuronal maturation/differentiation.

Coordinated induction of two unrelated glucose-regulated protein genes by a calcium ionophore: Human BiPGRP78 and GAPDH

The induction of human BiP/GRP78 and GAPDH protein genes by the calcium ionophore A23187 was determined. Steady-state levels of mRNA for both the glucose starvation-responsive BiP/GRP78 gene and the glucose-responsive GAPDH gene were dramatically induced in a variety of human cells. There is a homologous palindromatic sequence GCCGTTAACGGC in the active promoter region of the two genes that is known to be required for the induction of mammalian BiP/GRP78 by A23187. The evidence confirms in general the function of this element in the regulation of calcium-associated gene activity.

The spinal muscular atrophy disease protein SMN is linked to the Golgi network.

Proximal spinal muscular atrophy (SMA) is a neurodegenerative disorder caused by deficiency of the ubiquitous Survival of Motor Neuron (SMN) protein. SMN has been shown to be transported in granules along the axon and moved through cytoskeletal elements. However, the role and nature of SMN granules are still not well characterized. Here, using immunocytochemical methods and time-lapse studies we show that SMN granules colocalize with the Golgi apparatus in motor neuron-like NSC34 cells. Electron microscopy clearly revealed that SMN granules are transported into the Golgi stack and aggregate in the trans-Golgi apparatus. SMN granules are characterized as either coated or un-coated and behave like regulated secretory granules. Treatment of cells with monensin to disrupt Golgi-mediated granule secretion decreased SMN expression in neurites and caused growth cone defects similar to those seen in SMN knockdown cells. Knockdown of Cop-α, the protein that coats vesicles transporting proteins between the Golgi compartments, caused SMN granule accumulation in the Golgi apparatus. In addition to the well-studied role of SMN in small nuclear ribonucleoprotein (SnRNP) assembly, this work links SMN granules with the Golgi network and thus sheds light on Golgi-mediated SMN granule transport.

Involvement of Gas7 along the ERK1/2 MAP kinase and SOX9 pathway in chondrogenesis of human marrow-derived mesenchymal stem cells

OBJECTIVE The growth-arrest-specific protein, Gas7, has been shown to be involved in reorganization of the cytoskeleton and for inducing changes in cell shape during cell differentiation. The goals of this study were to investigate the novel role of human Gas7 (hGas7) in chondrogenic differentiation of human mesenchymal stem cells (hMSCs) and to identify the relationship between hGas7, extracellular signal-regulated kinase (ERK1/2) and SOX9 in the chondrogenic pathway. METHODS Bone marrow-derived hMSCs were induced to undergo chondrogenic differentiation with transforming growth factor-beta1 (TGF-beta1) in an aggregate culture system. The expression of hGas7 and SOX9 and phosphorylation of ERK1/2 at multiple time points were investigated. Chondrogenic capacity was evaluated by the size of aggregates, by glycosaminoglycan content, and by type II collagen and proteoglycan deposition after interfering with expression of hGas7, ERK1/2 or SOX9. To delineate the functional role of these genes in chondrogenesis, inhibition of individual genes expression in hMSCs, by antisense oligonucleotides or interference RNA (siRNA), and the effect on chondrogenic differentiation were also investigated. RESULTS Treatment of hMSCs with TGF-beta1 resulted in a transient up-regulation of hGas7b, one of the hGas7 isoforms (day 3-day 5), a transient phosphorylation of ERK1/2 (0.5-4 h) and an up-regulation of SOX9 (2 h to day 14). Transient expression of hGas7b was also detected in hMSCs by reverse transcription-polymerase chain reaction at day 2 and day 3 following TGF-beta1 treatment. Interference with hGas7b production by hGas7b-specific antisense oligonucleotide or inhibition of p-ERK with PD98059, a specific inhibitor of ERK signaling pathway, or interference with SOX9 production by SOX9 siRNA all caused adverse effects of chondrogenic differentiation of hMSCs. Meanwhile, inhibition of p-ERK or SOX9 both blocked the expression of hGas7b. However, the p-ERK and SOX9 pathway was not affected by inhibition of hGas7b. CONCLUSION These results provide evidence that the transient expression of hGas7b, regulated by activation of ERK1/2 and SOX9 pathway, is essential for chondrogenic differentiation of hMSCs.

Unraveling new roles for minor components of the E. coli RNA degradosome

Endoribonuclease E, 3’-5’ exoribonuclease polynucleotide phosphorylase, RhlB RNA helicase and enolase form a multienzyme complex (the ”degradosome”) playing an important role in RNA processing and decay in Escherichia coli. Although a number of proteins that occasionally co-purify with the E. coli degradosome in non-stoichiometric amounts were initially viewed as insignificant constituents of this complex, recent studies suggest that, in fact, some of these copurifying proteins are involved in modulation of degradosome composition, activity or specificity during bacterial adaptation to changing environments. Here we briefly review these findings and discuss their implications for understanding the multifaceted mechanisms controlling degradosome functions in vivo.