Current research on viral proteins that interact with fibrillarin.

The nucleolus is a multifunctional nuclear domain primarily dedicated to ribosome biogenesis. Certain viruses developed strategies to manipulate host nucleolar proteins to facilitate their replication by modulating ribosomal RNA (rRNA) processing. This association interferes with nucleolar functions resulting in overactivation or arrest of ribosome biogenesis, induction or inhibition of apoptosis, and affecting stress response. The nucleolar protein fibrillarin (FBL) is an important target of some plant and animal viruses. FBL is an essential and highly conserved S-adenosyl methionine (SAM) dependent methyltransferase, capable of rRNA degradation by its intrinsically disordered region (IDR), the glycine/arginine-rich (GAR) domain. It forms a ribonucleoprotein complex that directs 2'-O-methylations in more than 100 sites of pre-rRNAs. It is involved in multiple cellular processes, including initiation of transcription, oncogenesis, and apoptosis, among others. The interaction with animal viruses, including human viruses, triggered its redistribution to the nucleoplasm and cytoplasm, interfering with its role in pre-rRNA processing. Viral-encoded proteins with IDRs as nucleocapsids, matrix, Tat protein, and even a viral snoRNA, can associate with FBL, forcing the nucleolar protein to undergo atypical functions. Here we review the molecular mechanisms employed by animal and human viruses to usurp FBL functions and the effect on cellular processes, particularly in ribosome biogenesis.

Structural studies of human fission protein FIS1 reveal a dynamic region important for GTPase DRP1 recruitment and mitochondrial fission.

Fission protein 1 (FIS1) and dynamin-related protein 1 (DRP1) were initially described as being evolutionarily conserved for mitochondrial fission, yet in humans the role of FIS1 in this process is unclear and disputed by many. In budding yeast where Fis1p helps to recruit the DRP1 ortholog from the cytoplasm to mitochondria for fission, an N-terminal "arm" of Fis1p is required for function. The yeast Fis1p arm interacts intramolecularly with a conserved tetratricopeptide repeat core and governs in vitro interactions with yeast DRP1. In human FIS1, NMR and X-ray structures show different arm conformations, but its importance for human DRP1 recruitment is unknown. Here, we use molecular dynamics simulations and comparisons to experimental NMR chemical shifts to show the human FIS1 arm can adopt an intramolecular conformation akin to that observed with yeast Fis1p. This finding is further supported through intrinsic tryptophan fluorescence and NMR experiments on human FIS1 with and without the arm. Using NMR, we observed the human FIS1 arm is also sensitive to environmental changes. We reveal the importance of these findings in cellular studies where removal of the FIS1 arm reduces DRP1 recruitment and mitochondrial fission similar to the yeast system. Moreover, we determined that expression of mitophagy adapter TBC1D15 can partially rescue arm-less FIS1 in a manner reminiscent of expression of the adapter Mdv1p in yeast. These findings point to conserved features of FIS1 important for its activity in mitochondrial morphology. More generally, other tetratricopeptide repeat-containing proteins are flanked by disordered arms/tails, suggesting possible common regulatory mechanisms.

SAP130 and CSN1 interact and regulate male gametogenesis in Arabidopsis thaliana.

COP9 signalosome (CSN) is a nuclear complex composed of eight distinct subunits that governs vast developmental processes in Arabidopsis thaliana (L.) Heynh. The null alleles of csn mutants display pleiotropic phenotypes that result in seedling lethality. To date, several partially complemented transgenic plants, expressing the particular CSN subunit in its corresponding null mutant allele, were utilized to bypass seedling lethality and investigate CSN regulation at later stages of development. One such transgenic plant corresponding to CSN1 subunit, fus6/CSN1-3-4, accumulates wild-type level of CSN1 and displays normal plant architecture at vegetative stage. Here we show through histological analyses that fus6/CSN1-3-4 plants display impairment of pollen development at the bicellular stage. This defect is identical to that observed in RNAi plants of SAP130, encoding a subunit of the multiprotein splicing factor SF3b. We further dissected the previously reported interaction between CSN1 and SAP130, to reveal that approximately 100 amino-acid residues located at the N-terminal end of CSN1 (CSN1NN) were essential for this interaction. In silico structure modeling demonstrated that CSN1NN could swing out towards SAP130 to dock onto its Helical Insertion protruding from the structure. These results support our model that CSN1 embeds itself within CSN protein complex through its C-terminal half and reaches out to targets through its N-terminal portion of the protein. Taken together, this is the first report to document the identical loss-of-function phenotypes of CSN1 and SAP130 during male gametogenesis. Thus, we propose that SAP130 and CSN1 coordinately regulate development of male reproductive organs.

Salt-Dependent Modulation of the RNA Chaperone Activity of RNA-Binding Protein La.

It is well established that the RNA-binding protein La has RNA chaperone activity. Recent work suggests that the La protein has two distinct RNA chaperone domains (RCD-A and RCD-B) assisting structural changes in diverse groups of RNA molecules such as RNA polymerase III transcripts (e.g., pre-tRNA, U6 snRNA), cellular messenger, and viral RNAs. In this protocol we focus on the RNA chaperone domain RCD-B, which is located in the carboxy-terminal domain of La. It has been shown that this RNA chaperone domain assists structural changes in predicted RNA hairpins folded in the 5'-untranslated regions of cyclin D1 and Bcl2 mRNAs. Besides RNA helicases, which are implicated in melting RNA hairpin structures in an ATP-dependent manner, RNA chaperones fulfil a similar function in an ATP-independent manner. Aiming to study the RNA chaperon activity of La, we established a La-dependent molecular beacon-based RNA chaperone assay and systematically tested the various salt conditions. Herein we describe the assay format and design to study the salt dependency of RNA chaperones. This protocol can be easily adapted to test the RNA chaperone activity of other RNA-binding proteins and to optimize assay conditions.

Point mutation A394E in the central intrinsic disordered region of Rna14 leads to chromosomal instability in fission yeast.

Accurate chromosomal segregation is crucial for the maintenance of genomic integrity. Rna14 is a major component of the yeast pre-mRNA 3'-end processing factor, the cleavage factor IA complex, and is involved in cleavage and polyadenylation of mRNA in the nucleus. Rna14 is also essential for the maintenance of genomic integrity in fission yeast Schizosaccharomyces pombe. In the present study, we report that a non-homologous mutation, A394E that is present in the central intrinsic disordered region of Rna14 leads to chromosomal instability in fission yeast. This mutation was shown to disrupt chromosome segregation and 3'-end maturation, and also affects the pre-mRNA splicing in vivo at non-permissive temperatures. We observed that a significant part of Rna14 is intrinsically disordered, that includes the N- and C-terminal of Rna14, as well as the central region containing the HAT repeats and the mutation within amino acid residues 372-435. These regions are crucial for the function of Rna14 as they are involved in the interaction of Rna14 with other proteins.