Note:

Note:

This literature study provides a comprehensive overview of the current knowledge of mRNA-based pharmaceuticals, technologies and applications.  mRNA-based pharmaceuticals have newly been bought into focus as a potential brand-new drug class and many believe that it has the potential to transform areas of medicine. The corona pandemic showed that mRNA vaccines could be created fast and safely which highlighted the unique potential of mRNA therapy. This resulted in a major investment in mRNA therapeutics and attracted attention to synthetic mRNA as a new drug modality. The sequence of nucleotides in synthetically produced (IVT) mRNA can easily be altered to produce different proteins in vivo. This leads to a broad range of therapeutical applications such as protein replacement therapy, immunotherapy, genome editing, cellular programming, and vaccines.  The disadvantages of the mRNA technique will be discussed; primarily the low stabilization in vivo and low target delivery, which have partly been solved today. By nucleobase modifications the stabilization can be improved and by delivery systems an increased target delivery can be achieved.  The clinical promise of mRNA therapeutics and vaccines is crystal clear, however, their novelty brings new manufacturing challenges. Technologies and strategies to address these challenges will be concluded in this paper.

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RNA maturation involves several steps prior to export of the mRNA out of the nucleus and translation in the cytoplasm. PremRNA 3’end processing is one of such steps, and comprises the endonucleolytic cleavage and polyadenylation of the 3’end of the premRNA. These two steps involve more than 14 processing factors that coordinate multiple proteinprotein and proteinRNA interactions necessary to coordinate efficient cleavage and polyadenylation. To date, many of these interactions have been investigated biochemically and require additional structural characterization both to confirm and highlight key residues involved in substrate contacts. Further structural characterization will also open investigation into the mechanism of 3’end processing by providing structural insight into the coordination of multiple binding components. The cleavage factor Im, CF Im, is a component of the 3’end processing machinery and plays an important role early, during endonucleolytic cleavage, and additionally to increase polyadenylation efficiency and regulate poly(A) site recognition. CF Im is composed of a small 25 kDa subunit, CF Im25, and a large, either 58 kDa, 68 kDa, or 72 kDa subunit. The 25 kDa subunit of CF Im interacts with both the RNA and other processing factors such as the poly(A) polymerase, Clp1, and the larger subunit of CF Im. It is our goal to crystallize CF Im25 alone and in complex with one of its interacting partners to better understand CF Im25 contributions to premRNA 3’end processing. The structural investigation of CF Im25 and its binding partners has accomplished four major objectives: 1) Characterized the crystal structure of CF Im25 alone and bound to diadenosine tetraphosphate, 2) Provided insight into the oligomeric state of the CF Im complex, 3) Determined the binding properties of the Nudix domain of CF Im25 and its function in 3’end processing, 4) Further characterize the interactions between CF Im25 and PAP, CF Im68, and Clp1. These results demonstrate CF Im25 is a dimer both in solution and in the crystal suggesting that it is likely to be a dimer in the CF Im complex. The nucleotide binging capability of CF Im25 has no apparent role in 3’end processing in vitro but may provide a function outside of 3’end processing or may directly be involved in RNA recognition. The additional investigation of complex interactions with the 25 kDa subunit of CF Im25 suggests that although these factors interact during the 3’end processing event additional mechanisms may play a role in stabilizing those interactions.

Vts1 is a member of the Smaug protein family, a group of sequence-specific RNA-binding proteins that regulate mRNA translation and degradation by binding to consensus stem-loop structures in target mRNAs. Using RNA reporters that recapitulate Vts1-mediated decay in vivo as well as endogenous mRNA transcripts, I show that Vts1 regulates the degradation of target mRNAs in Saccharomyces cerevisiae. In Chapter Two, I focus on the mechanism of Vts1-mediated mRNA decay. I demonstrate that Vts1 initiates mRNA degradation through deadenylation mediated by the Ccr4-Pop2-Not deadenylase complex. I also show that Vts1 interacts with the Ccr4-Pop2-Not deadenylase complex suggesting that Vts1 recruits the deadenylase machinery to target mRNAs, resulting in transcript decay. Following poly(A) tail removal, Vts1 target transcripts are decapped and subsequently degraded by the 5’-to-3’ exonuclease Xrn1. Taken together these data suggest a mechanism of mRNA degradation that involves recruitment of the Ccr4-Pop2-Not deadenylase to target mRNAs. Previous work in Drosophila melanogaster demonstrated that Smg interacts with the Ccr4-Pop2-Not complex to regulate mRNA stability, suggesting Smaug family members employ a conserved mechanism of mRNA decay. In Drosophila, Smg also regulates mRNA translation through a separate mechanism involving the eIF4E-binding protein Cup. In Chapter Three, I identify the eIF4E-associated protein Eap1 as a component of Vts1-mediated mRNA decay in yeast. Interestingly Cup and Eap1 share no significant homology outside of the seven amino acid eIF4E-binding motif. In eap1 cells mRNAs accumulate as deadenylated capped species, suggesting that Eap1 stimulates mRNA decapping. I demonstrate that the Eap1 eIF4E-binding motif is required for efficient degradation of Vts1 target mRNAs and that this motif enables Eap1 to mediate an interaction between Vts1 and eIF4E. Together these data suggest Vts1 influences multiple steps in the mRNA decay pathway through interactions with the Ccr4-Pop2-Not deadenylase and the decapping activator Eap1.

Vts1 is a member of the Smaug protein family, a group of sequence-specific RNA-binding proteins that regulate mRNA translation and degradation by binding to consensus stem-loop structures in target mRNAs. Using RNA reporters that recapitulate Vts1-mediated decay in vivo as well as endogenous mRNA transcripts, I show that Vts1 regulates the degradation of target mRNAs in Saccharomyces cerevisiae. In Chapter Two, I focus on the mechanism of Vts1-mediated mRNA decay. I demonstrate that Vts1 initiates mRNA degradation through deadenylation mediated by the Ccr4-Pop2-Not deadenylase complex. I also show that Vts1 interacts with the Ccr4-Pop2-Not deadenylase complex suggesting that Vts1 recruits the deadenylase machinery to target mRNAs, resulting in transcript decay. Following poly(A) tail removal, Vts1 target transcripts are decapped and subsequently degraded by the 5’-to-3’ exonuclease Xrn1. Taken together these data suggest a mechanism of mRNA degradation that involves recruitment of the Ccr4-Pop2-Not deadenylase to target mRNAs. Previous work in Drosophila melanogaster demonstrated that Smg interacts with the Ccr4-Pop2-Not complex to regulate mRNA stability, suggesting Smaug family members employ a conserved mechanism of mRNA decay. In Drosophila, Smg also regulates mRNA translation through a separate mechanism involving the eIF4E-binding protein Cup. In Chapter Three, I identify the eIF4E-associated protein Eap1 as a component of Vts1-mediated mRNA decay in yeast. Interestingly Cup and Eap1 share no significant homology outside of the seven amino acid eIF4E-binding motif. In eap1 cells mRNAs accumulate as deadenylated capped species, suggesting that Eap1 stimulates mRNA decapping. I demonstrate that the Eap1 eIF4E-binding motif is required for efficient degradation of Vts1 target mRNAs and that this motif enables Eap1 to mediate an interaction between Vts1 and eIF4E. Together these data suggest Vts1 influences multiple steps in the mRNA decay pathway through interactions with the Ccr4-Pop2-Not deadenylase and the decapping activator Eap1.

Osteoblastic cells respond to mechanical stimuli with alterations in proliferation and/or phenotypic expression. In some cases, these responses occur within only a few applications of stimuli (i.e. 'cycle-dependent trigger response') rather than in a dose-dependent manner. To explore potential mechanisms of the cycle dependent trigger response, we raised the following questions: (1) Does strain of bone cells alter gene expression; if so, how quickly does it occur and how long does it last? (2) Are alterations in message level strain magnitude dependent? (3) Are alterations in steady-state message levels cycle dependent? Cultures were evaluated for osteocalcin mRNA one week following a daily stretch application at four stretch magnitudes and four cycle numbers and compared to nonstretched controls. Steady state mRNA message was ascertained prior to and at 10, 20, 30, 60, 120, 180, and 240 min following initiation of stretch. Following mRNA isolation, first strand cDNA synthesis was performed and fluorometrically quantitated. A reverse transcriptase based PCR (RT-PCR) approach allowed assessment of osteocalcin mRNA levels from microcultures (50,000 cells per 10 μl culture or 5000 cells mm2) of rat calvarial osteoblasts. Optimized PCR was performed using primers to the bone specific protein, osteocalcin (OC) and two 'housekeeping' genes, β-actin and GAP-DH. PCR products were separated on 4% agarose gels and band intensities digitized with relative quantitation based on internal standards in each gel. The lowest magnitude of stretch (-1 KPa) at 1800 cycles per day reproducibly depressed message for osteocalcin, but not β-actin when assayed immediately following the cessation of strain application. By three hours following the initiation of stretch, message levels returned to control values. At the time of stretch cessation, the 1800 cycle stretch regimen diminished (p < 0.0001) steady-state osteocalcin message independently of the four stretch magnitudes. Stretch for 300 cycles failed to depress (p = 0.05) osteocalcin message cultures at any time, but 600 cycles depressed message by 30 min. By one and two hours, cultures stretched 600, 900, and 1800 cycles showed similar levels of message depression. Four hours following the initiation of stretch, message levels returning to nonstrained levels in all groups. We conclude that alterations in cell response to strain are in part mediated by gene expression, that alterations last 3-4 h in this system, and that the message mechanism itself exhibits a triggerresponse dependency to cycle number.