investigation-protein-recruitment-to-dna-lesions-using-405-nm-laser

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand ...

Bcl-x is a member of the Bcl-2 family of proteins that are key regulators of apoptosis. The Bcl-x pre-mRNA is alternatively spliced to yield Bcl-x(S) and Bcl-x(L), two isoforms that have been associated, respectively, with the promotion and the prevention of apoptosis. We have investigated some of the elements and factors involved in the production of these two splice variants. Deletion mutagenesis using a human Bcl-x minigene identifies two regions in exon 2 that modulate Bcl-x 5'-splice site selection in human HeLa cells. One region (B3) is located upstream of the Bcl-x(L) 5'-splice site and enforces Bcl-x(L) production in cells and splicing extracts. The other region (B2) is located immediately downstream of the 5'-splice site of Bcl-x(S) and favors Bcl-x(S) production in vivo and in vitro. A 30-nucleotide G-rich element (B2G) is responsible for the activity of the B2 element. We show that recombinant heterogeneous nuclear ribonucleoprotein (hnRNP) F and H proteins bind to B2G, and mutating the G stretches abolishes binding. Moreover, the addition of hnRNP F to a HeLa extract improved the production of the Bcl-x(S) variant in a manner that was dependent on the integrity of the G stretches in B2G. Consistent with the in vitro results, small interfering RNA-mediated RNA interference targeting hnRNP F and H decreased the Bcl-x(S)/Bcl-x(L) ratio of plasmid-derived and endogenously produced Bcl-x transcripts. Our results document a positive role for the hnRNP F/H proteins in the production of the proapoptotic regulator Bcl-x(S.).

Heterogeneous nuclear ribonucleoprotein F/H proteins modulate the alternative splicing of the apoptotic mediator Bcl-x.

We previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo. The tailed oligonucleotides that were used interfere with splicing because they contain a portion complementary to sequences immediately upstream of the target 5' splice site combined with a non-hybridizing 5' tail carrying binding sites for the hnRNP A1/A2 proteins. In the present study, we have tested the inhibitory activity of RNA oligonucleotides carrying different tail structures.

Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals.

The alternative splicing of Bcl-x generates the proapoptotic Bcl-x(S) protein and the antiapoptotic isoform Bcl-x(L). Bcl-x splicing is coupled to signal transduction, since ceramide, hormones, and growth factors alter the ratio of the Bcl-x isoforms in different cell lines. Here we report that the protein kinase C (PKC) inhibitor and apoptotic inducer staurosporine switches the production of Bcl-x towards the x(S) mRNA isoform in 293 cells. The increase in Bcl-x(S) elicited by staurosporine likely involves signaling events that affect splicing decisions, because it requires active transcription and no new protein synthesis and is independent of caspase activation. Moreover, the increase in Bcl-x(S) is reproduced with more specific inhibitors of PKC. Alternative splicing of the receptor tyrosine kinase gene Axl is similarly affected by staurosporine in 293 cells. In contrast to the case for 293 cells, PKC inhibitors do not influence the alternative splicing of Bcl-x and Axl in cancer cell lines, suggesting that these cells have sustained alterations that uncouple splicing decisions from PKC-dependent signaling. Using minigenes, we show that an exonic region located upstream of the Bcl-x(S) 5' splice site is important to mediate the staurosporine shift in Bcl-x splicing. When transplanted to other alternative splicing units, portions of this region confer splicing modulation and responsiveness to staurosporine, suggesting the existence of factors that couple splicing decisions with PKC signaling.

Protein kinase C-dependent control of Bcl-x alternative splicing.

Most human genes produce multiple mRNA isoforms through alternative splicing. However, the biological relevance of most splice variants remains unclear. In this study, we evaluated the functional impact of alternative splicing in cancer cells. We modulated the splicing pattern of 41 cancer-associated splicing events and scored the effects on cell growth, viability and apoptosis, identifying three isoforms essential for cell survival. Specifically, changing the splicing pattern of the spleen tyrosine kinase gene (SYK) impaired cell-cycle progression and anchorage-independent growth. Notably, exposure of cancer cells to epithelial growth factor modulated the SYK splicing pattern to promote the pro-survival isoform that is associated with cancer tissues in vivo. The data suggest that splicing of selected genes is specifically modified during tumor development to allow the expression of isoforms that promote cancer cell survival.

Alternative splicing of SYK regulates mitosis and cell survival.

Several apoptotic regulators, including Bcl-x, are alternatively spliced to produce isoforms with opposite functions. We have used an RNA interference strategy to map the regulatory landscape controlling the expression of the Bcl-x splice variants in human cells. Depleting proteins known as core (Y14 and eIF4A3) or auxiliary (RNPS1, Acinus, and SAP18) components of the exon junction complex (EJC) improved the production of the proapoptotic Bcl-x(S) splice variant. This effect was not seen when we depleted EJC proteins that typically participate in mRNA export (UAP56, Aly/Ref, and TAP) or that associate with the EJC to enforce nonsense-mediated RNA decay (MNL51, Upf1, Upf2, and Upf3b). Core and auxiliary EJC components modulated Bcl-x splicing through different cis-acting elements, further suggesting that this activity is distinct from the established EJC function. In support of a direct role in splicing control, recombinant eIF4A3, Y14, and Magoh proteins associated preferentially with the endogenous Bcl-x pre-mRNA, interacted with a model Bcl-x pre-mRNA in early splicing complexes, and specifically shifted Bcl-x alternative splicing in nuclear extracts. Finally, the depletion of Y14, eIF4A3, RNPS1, SAP18, and Acinus also encouraged the production of other proapoptotic splice variants, suggesting that EJC-associated components are important regulators of apoptosis acting at the alternative splicing level.

Proteins associated with the exon junction complex also control the alternative splicing of apoptotic regulators.

Pre-mRNA alternative splicing is modified in cancer, but the origin and specificity of these changes remain unclear. Here, we probed ovarian tumors to identify cancer-associated splicing isoforms and define the mechanism by which splicing is modified in cancer cells. Using high-throughput quantitative PCR, we monitored the expression of splice variants in laser-dissected tissues from ovarian tumors. Surprisingly, changes in alternative splicing were not limited to the tumor tissues but were also found in the tumor microenvironment. Changes in the tumor-associated splicing events were found to be regulated by splicing factors that are differentially expressed in cancer tissues. Overall, ∼20% of the alternative splicing events affected by the down-regulation of the splicing factors QKI and RBFOX2 were altered in the microenvironment of ovarian tumors. Together, our results indicate that the tumor microenvironment undergoes specific changes in alternative splicing orchestrated by a limited number of splicing factors.

Tumor microenvironment-associated modifications of alternative splicing.

Small nucleolar RNAs (snoRNAs) are among the first discovered and most extensively studied group of small non-coding RNA. However, most studies focused on a small subset of snoRNAs that guide the modification of ribosomal RNA. In this study, we annotated the expression pattern of all box C/D snoRNAs in normal and cancer cell lines independent of their functions. The results indicate that C/D snoRNAs are expressed as two distinct forms differing in their ends with respect to boxes C and D and in their terminal stem length. Both forms are overexpressed in cancer cell lines but display a conserved end distribution. Surprisingly, the long forms are more dependent than the short forms on the expression of the core snoRNP protein NOP58, thought to be essential for C/D snoRNA production. In contrast, a subset of short forms are dependent on the splicing factor RBFOX2. Analysis of the potential secondary structure of both forms indicates that the k-turn motif required for binding of NOP58 is less stable in short forms which are thus less likely to mature into a canonical snoRNP. Taken together the data suggest that C/D snoRNAs are divided into at least two groups with distinct maturation and functional preferences.

Identification of discrete classes of small nucleolar RNA featuring different ends and RNA binding protein dependency.

Considering the medical, biotechnological, and economical importance of actinobacteria, there is a continuous need to improve the tools for genetic engineering of a broad range of these microorganisms. Intergeneric conjugation has proven to be a valuable yet imperfect tool for this purpose. The natural resistance of many actinomycetes to nalidixic acid (Nal) is generally exploited to eliminate the sensitive Escherichia coli donor strain following conjugation. Nevertheless, Nal can delay growth and have other unexpected effects on the recipient strain. To provide an improved alternative to antibiotics, we propose a postconjugational counterselection using a diaminopimelic acid (DAP) auxotrophic donor strain. The DAP-negative phenotype was obtained by introducing a dapA deletion into the popular methylase-negative donor strain E. coli ET12567/pUZ8002. The viability of ET12567 and its ΔdapA mutant exposed to DAP deprivation or Nal selection were compared in liquid pure culture and after mating with Streptomyces coelicolor. Results showed that death of the E. coli ΔdapA Nal-sensitive donor strain occurred more efficiently when subjected to DAP deprivation than when exposed to Nal. Our study shows that postconjugational counterselection based on DAP deprivation circumvents the use of antibiotics and will facilitate the transfer of plasmids into actinomycetes with high biotechnological potential, yet currently not accessible to conjugative techniques.

A diaminopimelic acid auxotrophic Escherichia coli donor provides improved counterselection following intergeneric conjugation with actinomycetes.

Colonization of plant roots by Bacillus subtilis is mutually beneficial to plants and bacteria. Plants can secrete up to 30% of their fixed carbon via root exudates, thereby feeding the bacteria, and in return the associated B. subtilis bacteria provide the plant with many growth-promoting traits. Formation of a biofilm on the root by matrix-producing B. subtilis is a well-established requirement for long-term colonization. However, we observed that cells start forming a biofilm only several hours after motile cells first settle on the plant. We also found that intact chemotaxis machinery is required for early root colonization by B. subtilis and for plant protection. Arabidopsis thaliana root exudates attract B. subtilis in vitro, an activity mediated by the two characterized chemoreceptors, McpB and McpC, as well as by the orphan receptor TlpC. Nonetheless, bacteria lacking these chemoreceptors are still able to colonize the root, suggesting that other chemoreceptors might also play a role in this process. These observations suggest that A. thaliana actively recruits B. subtilis through root-secreted molecules, and our results stress the important roles of B. subtilis chemoreceptors for efficient colonization of plants in natural environments. These results demonstrate a remarkable strategy adapted by beneficial rhizobacteria to utilize carbon-rich root exudates, which may facilitate rhizobacterial colonization and a mutualistic association with the host.

Article	Year
Heterogeneous nuclear ribonucleoprotein F/H proteins modulate the alternative splicing of the apoptotic mediator Bcl-x. The Journal of biological chemistry\| PubMed ID: 15837790	2005
Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals. BMC biotechnology\| PubMed ID: 16412215	2006
Protein kinase C-dependent control of Bcl-x alternative splicing. Molecular and cellular biology\| PubMed ID: 17923691	2007
Alternative splicing of SYK regulates mitosis and cell survival. Nature structural & molecular biology\| PubMed ID: 21552259	2011
Proteins associated with the exon junction complex also control the alternative splicing of apoptotic regulators. Molecular and cellular biology\| PubMed ID: 22203037	2012
Tumor microenvironment-associated modifications of alternative splicing. RNA (New York, N.Y.)\| PubMed ID: 24335142	2014
Identification of discrete classes of small nucleolar RNA featuring different ends and RNA binding protein dependency. Nucleic acids research\| PubMed ID: 25074380	2014
A diaminopimelic acid auxotrophic Escherichia coli donor provides improved counterselection following intergeneric conjugation with actinomycetes. Canadian journal of microbiology\| PubMed ID: 26166710	2015
Bacillus subtilis Early Colonization of Arabidopsis thaliana Roots Involves Multiple Chemotaxis Receptors. mBio\| PubMed ID: 27899502	2016

Daniel Garneau

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