Fungal CSL transcription factors

<p>Abstract</p> <p>Background</p> <p>The CSL (CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1) transcription factor family members are well-known components of the transmembrane receptor Notch signaling pathway, which plays a critical role in metazoan development. They function as context-dependent activators or repressors of transcription of their responsive genes, the promoters of which harbor the GTG(G/A)GAA consensus elements. Recently, several studies described Notch-independent activities of the CSL proteins.</p> <p>Results</p> <p>We have identified putative CSL genes in several fungal species, showing that this family is not confined to metazoans. We have analyzed their sequence conservation and identified the presence of well-defined domains typical of genuine CSL proteins. Furthermore, we have shown that the candidate fungal protein sequences contain highly conserved regions known to be required for sequence-specific DNA binding in their metazoan counterparts. The phylogenetic analysis of the newly identified fungal CSL proteins revealed the existence of two distinct classes, both of which are present in all the species studied.</p> <p>Conclusion</p> <p>Our findings support the evolutionary origin of the CSL transcription factor family in the last common ancestor of fungi and metazoans. We hypothesize that the ancestral CSL function involved DNA binding and Notch-independent regulation of transcription and that this function may still be shared, to a certain degree, by the present CSL family members from both fungi and metazoans.</p

Secondary structure is required for 3′ splice site recognition in yeast

Higher order RNA structures can mask splicing signals, loop out exons, or constitute riboswitches all of which contributes to the complexity of splicing regulation. We identified a G to A substitution between branch point (BP) and 3′ splice site (3′ss) of Saccharomyces cerevisiae COF1 intron, which dramatically impaired its splicing. RNA structure prediction and in-line probing showed that this mutation disrupted a stem in the BP-3′ss region. Analyses of various COF1 intron modifications revealed that the secondary structure brought about the reduction of BP to 3′ss distance and masked potential 3′ss. We demonstrated the same structural requisite for the splicing of UBC13 intron. Moreover, RNAfold predicted stable structures for almost all distant BP introns in S. cerevisiae and for selected examples in several other Saccharomycotina species. The employment of intramolecular structure to localize 3′ss for the second splicing step suggests the existence of pre-mRNA structure-based mechanism of 3′ss recognition

Fission yeast CSL proteins function as transcription factors.

BACKGROUND: Transcription factors of the CSL (CBF1/RBP-Jk/Suppressor of Hairless/LAG-1) family are key regulators of metazoan development and function as the effector components of the Notch receptor signalling pathway implicated in various cell fate decisions. CSL proteins recognize specifically the GTG[G/A]AA sequence motif and several mutants compromised in their ability to bind DNA have been reported. In our previous studies we have identified a number of novel putative CSL family members in fungi, organisms lacking the Notch pathway. It is not clear whether these represent genuine CSL family members. METHODOLOGY/PRINCIPAL FINDINGS: Using a combination of in vitro and in vivo approaches we characterized the DNA binding properties of Cbf11 and Cbf12, the antagonistic CSL paralogs from the fission yeast, important for the proper coordination of cell cycle events and the regulation of cell adhesion. We have shown that a mutation of a conserved arginine residue abolishes DNA binding in both CSL paralogs, similar to the situation in mouse. We have also demonstrated the ability of Cbf11 and Cbf12 to activate gene expression in an autologous fission yeast reporter system. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the fission yeast CSL proteins are indeed genuine family members capable of functioning as transcription factors, and provide support for the ancient evolutionary origin of this important protein family

The N-termini of Cbf11 and Cbf12 are important for their nuclear localization.

<p>(<b>A</b>) Schematic representation of all Cbf11 and Cbf12 variants used in this study. The N-terminal Rel homology region ('RHR-N', green), beta trefoil domain ('BTD', red), and position of the point mutation affecting DNA binding activity are indicated. (<b>B</b>) A western blot confirming that all N-terminally EGFP-tagged CSL variants are expressed and produce a band of the expected size. The anti-PSTAIRE (Cdc2) western blot serves as a loading control. (<b>C</b>) All EGFP-CSL derivatives localize to the nucleus with the exception of variants devoid of the whole unstructured N-terminal region. Scale bar is 10 µm. The bottom panel shows longitudinal fluorescence intensity profiles of the cells marked with an asterisk.</p

Oligonucleotides used in this study.

a<p>'fwd'–forward primer; 'rev'–reverse primer.</p

Cbf11 binds to and activates a reporter gene <i>in vivo</i>.

<p>(<b>A</b>) Schematic representation of expression reporter plasmids with the β-galactosidase gene under the control of a minimal promoter with three copies of either the canonical ('RBP') or mutated ('DEL2') CSL response element (not to scale). (<b>B</b>) β-galactosidase activity in wild-type and CSL deletion mutant strains harbouring the RBP reporter plasmid. Activation of the reporter is dependent on endogenous Cbf11 but not Cbf12. (<b>C</b>) β-galactosidase activity in the <i>Δcbf11 Δcbf12</i> strain harbouring the RBP reporter plasmid and overexpressing the indicated CSL protein variants (HA-tagged) from a plasmid. The presence of high levels of both Cbf11 and Cbf12 activates, to a different degree, the RBP reporter. This activation is abolished in both CSL proteins by the 'DBM' point mutation in the BTD domain. (<b>D</b>) β-galactosidase activity in strains with chromosomally TAP-tagged Cbf11 or Cbf12 harbouring either the RBP or mutated DEL2 reporter plasmid. Both strains show RBP reporter activation similar to the untagged wild-type strain in (B) and no activation of the DEL2 reporter with mutated CSL binding sites. (<b>E, F</b>) Western blots of cell extracts from strains used in (C) and (D), respectively, confirming that all N-terminally HA-tagged and C-terminally TAP-tagged CSL variants were expressed properly. (<b>G</b>) Chromatin immunoprecipitation of TAP-tagged Cbf11 and Cbf12 from strains described in (D). Cbf11 binds strongly the promoter region of the RBP reporter but not the mutated DEL2 reporter. No binding to either reporter could be detected for Cbf12. All data represent mean values±standard deviations from at least three independent experiments.</p

Fission yeast strains used in this study.

<p>Fission yeast strains used in this study.</p

Mutation of a conserved arginine residue abolishes CSL DNA binding activity <i>in vitro</i>.

<p>(<b>A</b>) Substitution of the arginine residue at position 318 with histidine ('DBM') or a complete removal of the unstructured N-terminus abrogate the binding of Cbf11 to a DNA probe ('RBP') with the canonical CSL response element in an EMSA experiment. (<b>B</b>) The EMSA band corresponding to the DNA binding activity of the full-length non-mutated Cbf11 can be competed with an excess of the unlabelled RBP probe but not with the mutated DEL2 probe. (<b>C</b>) Full-length non-mutated Cbf12 displays very weak but specific DNA binding activity towards the RBP probe in EMSA. The Cbf12 band shift can be competed with an excess of the unlabelled RBP probe but not with the mutated DEL2 probe. No DNA binding activity was observed when the arginine residue at position 644 was substituted with histidine ('DBM'). A lane with low amounts of Cbf12(Δ1-394), previously shown to bind the RBP probe <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059435#pone.0059435-Pevorovsk2" target="_blank">[15]</a>, is shown for comparison (n.b., during native electrophoresis, protein migration speed is not simply proportional to protein mass since native charge and shape also play important roles). (<b>D</b>) Supershift experiments with GFP-tagged Cbf12 variants. For both full-length Cbf12 and Cbf12(Δ1-394) the addition of an anti-GFP antibody, but not an anti-TAP antibody, interferes with protein-DNA complex formation. 'w'–wells; 'p'–free probe. (<b>E, F, G</b>) Western blots of cell extracts from (A), (C) and (D), respectively, confirming that all N-terminally HA-tagged and GFP-tagged CSL variants were expressed properly and that equal amounts of cell extracts were used.</p