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In fission yeast lacking the telomere binding protein, Taz1, replication forks stall at telomeres, triggering deleterious downstream events. Strand invasion from one taz1Δ telomeric stalled fork to another on a separate (nonsister) chromosome leads to telomere entanglements, which are resolved in mitosis at 32°C; however, entanglement resolution fails at ≤20°C, leading to cold-specific lethality. Previously, we found that loss of the mitotic function of Rif1, a conserved DNA replication and repair factor, suppresses cold sensitivity by promoting resolution of entanglements without affecting entanglement formation. To understand the underlying pathways of mitotic entanglement resolution, we performed a series of genome-wide synthetic genetic array screens to generate a comprehensive list of genetic interactors of taz1 Δ and rif1 Δ. We modified a previously described screening method to ensure that the queried cells were kept in log phase growth. In addition to recapitulating previously identified genetic interactions, we find that loss of genes encoding components of the nuclear pore complex (NPC) promotes telomere disentanglement and suppresses taz1Δ cold sensitivity. We attribute this to more rapid anaphase midregion nuclear envelope (NE) breakdown in the absence of these NPC components. Loss of genes involved in lipid metabolism reverses the ability of rif1 + deletion to suppress taz1Δ cold sensitivity, again pinpointing NE modulation. A rif1 + separation-of-function mutant that specifically loses Rif1's mitotic functions yields similar genetic interactions. Genes promoting membrane fluidity were enriched in a parallel taz1+ synthetic lethal screen at permissive temperature, cementing the idea that the cold specificity of taz1Δ lethality stems from altered NE homeostasis. [ABSTRACT FROM AUTHOR]

We have previously shown that the Heat Shock Protein 90 (Hsp90) gene in G. lamblia is expressed from two ORFs localized 777 kb apart. The pre‐mRNAs transcribed from these ORFs are stitched by a trans‐splicing mechanism. Here, we provide mechanistic details of this process by reconstituting the reaction using in vitro synthesized pre‐mRNA substrates. Using RT‐PCR, northern blot and nanostring technology, we demonstrate that the in vitro synthesized pre‐mRNAs have the capability to self‐splice in the absence of nuclear proteins. Inhibition of the trans‐splicing reaction using a ssDNA oligo corresponding to a 26‐nucleotide complementary sequence confirmed their role in juxtapositioning the pre‐mRNA substrates during the self‐splicing reaction. Our study provides the first example of a self catalysed, trans‐splicing reaction in eukaryotes. [ABSTRACT FROM AUTHOR]

Background: Hsp90 from Giardia lamblia is expressed by splicing of two independently transcribed RNA molecules, coded by genes named HspN and HspC located 777 kb apart. The reasons underlying such unique trans-splicing based generation of GlHsp90 remain unclear. Principle Finding: In this study using mass-spectrometry we identify the sequence of the unique, junctional peptide contributed by the 5′ UTR of HspC ORF. This peptide is critical for the catalytic function of Hsp90 as it harbours an essential “Arg” in its sequence. We also show that full length GlHsp90 possesses all the functional hall marks of a canonical Hsp90 including its ability to bind and hydrolyze ATP. Using qRT-PCR as well as western blotting approach we find the reconstructed Hsp90 to be induced in response to heat shock. On the contrary we find GlHsp90 to be down regulated during transition from proliferative trophozoites to environmentally resistant cysts. This down regulation of GlHsp90 appears to be mechanistically linked to the encystation process as we find pharmacological inhibition of GlHsp90 function to specifically induce encystation. Significance: Our results implicate the trans-spliced GlHsp90 from Giardia lamblia to regulate an essential stage transition in the life cycle of this important human parasite. [ABSTRACT FROM AUTHOR]

Background: Hsp90 from Giardia lamblia is expressed by splicing of two independently transcribed RNA molecules, coded by genes named HspN and HspC located 777 kb apart. The reasons underlying such unique trans-splicing based generation of GlHsp90 remain unclear. Principle Finding: In this study using mass-spectrometry we identify the sequence of the unique, junctional peptide contributed by the 5′ UTR of HspC ORF. This peptide is critical for the catalytic function of Hsp90 as it harbours an essential “Arg” in its sequence. We also show that full length GlHsp90 possesses all the functional hall marks of a canonical Hsp90 including its ability to bind and hydrolyze ATP. Using qRT-PCR as well as western blotting approach we find the reconstructed Hsp90 to be induced in response to heat shock. On the contrary we find GlHsp90 to be down regulated during transition from proliferative trophozoites to environmentally resistant cysts. This down regulation of GlHsp90 appears to be mechanistically linked to the encystation process as we find pharmacological inhibition of GlHsp90 function to specifically induce encystation. Significance: Our results implicate the trans-spliced GlHsp90 from Giardia lamblia to regulate an essential stage transition in the life cycle of this important human parasite. [ABSTRACT FROM AUTHOR]