RNAs undergo different posttranscriptional chemical modifications, which affect their structural stability and functional diversity. RNA methylation is a very common type of post-transcriptional modification and is present in all domains of life: Archaea, Eukaryotes and Bacteria. Some of these methylations are catalyzed either by a RNA-protein complex or by stand-alone enzymes. The RNA-protein complex (Ribonucleoprotein complex) is comprised of a small RNA known as the guide RNA (Box C/D RNA) and core proteins (L7Ae, Nop5, and Fibrillarin). Box C/D RNAs contain conserved regions, called box C and box D near their 5’ and 3’ termini, respectively, and their imperfect copies called box C’ and box D’, internally. A short stretch of sequence between these Boxes are known as the guide/spacer regions, as the guide region helps in recruiting and positioning a specific target RNA for modification. Both in Archaea and Eukarya, box C and box D, as well as box C’ and box D’ together can form a structure called a Kink-turn (K-turn) that is characterized by a canonical Watson-Crick base-paired stem on one side, and a non-canonical stem on the other, separated by a 3-nucleotide loop. In Archaea box C’ and D’ can also form a K-loop, where the canonical stem of K-turn is replaced by a loop. Archaeal L7Ae binds first to the K-turn or K-loop and allows the recruitment of other proteins to form the complex. The presence of a unique box C/D RNA of Haloferax volcanii, called sR-tMet has been reported previously to guide the 2’-O-methylation of C34 in elongator pre-tRNAMet. Here we tried to characterize the structure-function relationship of this guide RNA under in vivo conditions. This RNA lacks a conventional K-turn or K-loop at its C’/D’ motif. We have created an H. volcanii strain that has a genomic deletion of sR-tMet. The sR-tMet gene is not essential for H. volcanii but this sR-tMet deleted strain lacks the 2’-O-methylation of C34 of its elongator tRNAMet. Unlike the close sR-tMet homologs (sR8 from Methanocaldococcus jannaschii and sR49 from Pyrococcus abyssi), the Box C’/D’ motif of sR-tMet is neither a K-turn nor a K-loop. The introduction of proper K-loop in the Box C’/D’ motif (sR-tMet with K-loop) abolished its Cm34 modification function in ΔsR-tMet strain. Direct interaction between L7Ae and the K-loop is not an absolute requirement for its function. However, disruption of the G/A and A/G pairing in Box C/D motif and Box D’ suggests the importance of these non-Watson crick base pairings in respect to sR-tMet’s function. Several other mutational studies have revealed that peculiar sR-tMet guide RNA from H. volcanii, behaves more like a Eukaryotic Box C/D RNA (where the K-loop is not required and presence of longer spacer length) than regular Archaeal one. Pseudouridine synthase 10 (Pus10) is the most recently identified Ψ synthase, found only in higher eukaryotes and Archaea. Archaeal Pus10 produces either tRNA Ψ55 or both tRNA Ψ54 and Ψ55 modifications. In Human, its Ψ synthase activity is not yet confirmed and interestingly it has been implicated in apoptosis. Herein for the first time we revealed that this putative RNA Ψ synthase protein, Human Pus10 (HuP10), translocates from the nucleus to the cytoplasm in TRAIL induced apoptosis. This nucleo-cytoplasmic movement of HuP10 occurs through the CRM1 mediated nuclear export pathway and Caspase 3 influences this movement. HuP10 also mediates crosstalk between the extrinsic and intrinsic pathways during TRAIL-induced apoptosis. Other than its involvement in apoptosis, we have also uncovered that HuP10 is involved in regulation of cell proliferation. Depletion (knockdown) of this protein in different cancer cell lines, promotes cell migration and anchorage-independent cell growth in the absence of any apoptotic stimulation.

RNA in all forms of life contain a myriad of post-transcriptional modifications. These modifications are important for processing and structural reasons, and includes 2’-O-methylation and pseudouridylation. Some of these modifications are the product of stand-alone proteins and others are the product of RNA-protein complexes.In eukaryotic and archaeal cells, the Box C/D ribonucleoprotein complex is one of the complexes responsible for 2’-O-methylation activity. In Archaea, this complex consists of the enzymatic protein, Fibrillarin, plus two other structural proteins, Nop5 and L7Ae, along with a guide RNA, which all come together to modify a specific target RNA sequence. A methyl group is added to the 2’ hydroxyl of the nucleotide’s ribose sugar. These modifications are found both in ribosomal RNA and tRNAs. This work focuses on the interactions between Nop5, Fibrillarin, and the guide RNA used in this complex. The objective was to identify important amino acid sequences in these proteins which are essential for the operation of the complex. The size of the archaeal Box C/D complex is also investigated using size chromatography.Similarly, Pus10 is one protein responsible for pseudouridylation in Archaea and eukaryotes. Pseudouridylation is the isomerization of uridine (U) to pseudouridine (Ψ). Pus10 is known to produce Ψ54 and Ψ55 in the TΨC loop of some archaeal and eukaryotic tRNAs. However, current research suggests it could have additional jobs in the cellular lifecycle, such as roles in apoptosis and the regulation of eukaryotic cell cycle. During mammalian cell apoptosis, Pus10 translocates from the nucleus to the cytoplasm and is believed to be involved in cytochrome c release. This is suspected to be related to Caspase-3’s role in apoptosis. Caspase-3 is activated in both the extrinsic and intrinsic apoptotic pathways. It has been shown that activation of the extrinsic apoptotic pathway via TRAIL affects the localization of Pus10. Here we test Pus10’s actions in cells under the effect of intrinsic apoptotic pathway compounds.Pus10 has also shown to have an effect on cell proliferation. Cells in which Pus10 has been depleted show increased growth rates. Here we investigate expression levels of proteins involved in cell cycle regulation, in both wild-type and Pus10-depleted cells, and attempt to compile a model of how Pus10 could be interacting with this system, using RNA-Seq, qPCR, and ChIP.SARS-CoV-2, also known as COVID-19, is a coronavirus that quickly became a pandemic in late 2019, early 2020. In the two years since then, the virus has undergone many mutations. By tracking the spread of these mutations at a national or regional level, by studying the spread pattern, we can make predictions, and possibly even prevent the next pandemic.