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In situ hybridization is used for detection of RNA expression when conservation of tissue architecture is important. Most in situ hybridization protocols are written for tissues from animals (i.e., rat) which can be harvested and preserved rapidly. In contrast, human tissue is more difficult to obtain, hence in situ hybridization experiments must frequently be performed with less than optimal tissue preservation. This procedure details hybridization of a radiolabeled single-stranded RNA probe (riboprobe) to complementary sequences of cellular RNA in human tissue sections. This method enables detection of rare mRNA species in specific cell types of human tissue, offering distinct advantages over other in situ methods due to increased sensitivity. In particular, we have found that UV cross-linking and ribonuclease treatment protocols need to be altered for human tissues to ensure successful results, making this protocol unique to those previously described. In situ hybridization experiments can be performed using either DNA or RNA probes. RNA probes are advantageous since they form stable hybrids, are single-stranded, have little or no reannealing during hybridization, and can be synthesized to high specific activity. RNA probes can be readily created utilizing SP6, T3, or T7 promoters in both sense and antisense orientations to provide non-specific (control) and specific probes. Disadvantages of RNA riboprobes include a tendency for RNA to stick non- selectively more than DNA, and degradation by RNase (hence strict adherence to RNase-free precautions is mandatory during most of the protocol). The following protocol includes: (1) preparation of human tissues (tissue fixation and sectioning are highlighted as critical for probe penetration, preservation of tissue architecture, retention of tissue RNA, and overall success); (2) generation of radiolabeled riboprobes (total incorporation of radionucleotide is important to increase sensitivity; 35S was chosen as a compromise between excellent sensitivity, cellular resolution, and required exposure times (compared with 32p or 3H); non-isotopic methods have not been tested in a side-by-side comparison with 35S in human tissues by us, but theoretically might offer faster exposure times while maintaining high resolution); (3) hybridization conditions (stringency, temperature, washes, tissue dehydration); and (4) sample visualization (application of photographic emulsion, developing, fixing, staining, and counterstaining of individual slides).

mRNA decay is an important cellular process that regulates gene expression and is tightly linked to the process of translation. Many studies have illustrated the link between mRNA turnover and translation, indicating that mRNA decay is a cytoplasmic event. In order to investigate further the link between translation and turnover, seven mutants in translation initiation factors were analyzed for their effect on mRNA decay, including: i) three mutant alleles of the PRT1 gene (prt1-1, prt1-26 and prt1-63), which encodes a subunit of elF3; ii) sui1-1, which encodes the smallest subunit of elF3; iii) sui2-1, which encodes elF2; iv) GCN2c, which encodes the elF2 kinase, and v) cdc33-42, a mutant in the cap binding protein elF4E. The results demonstrate that the prt1-1 mutation results in stabilization of nonsense containing mRNAs without affecting the half-lives of most other mRNAs, a phenotype similar to a upf1Δ strain. To identify substrates for the nonsense-mediated mRNA decay pathway, mRNA differential display analysis was performed using RNA prepared from prt1-1, PRT1, upf1Δ and UPF1 strains. Although the abundance of the HHF2 mRNA is increased in the mutant strains the half-life is unaffected. However, the mRNA half-life of the transcriptional regulator SPT10 was increased in the mutant strains indicating the SPT10 transcript is a substrate for the nonsense-mediated mRNA decay pathway. Further characterization of the SPT10 transcript showed that it is a substrate for this pathway because the initiator AUG is present in a poor translation initiation context which results in aberrant translation initiation. Finally, several other mRNAs, predicted to be substrates for the pathway based on the leaky scanning model, were subsequently shown to decay through this pathway. These transcripts included the REV7, STE50, and UBP7 mRNAs. The results from these experiments lay the groundwork for addressing the potential regulatory role of the nonsense-mediated mRNA decay pathway.

Splicing of mRNA precursors (pre-mRNA) comprises a series of ATP-dependent steps, the first of which is the stable binding of U2 snRNP at the pre-mRNA branchpoint. The basis of ATP use in splicing is not well understood. Several yeast splicing factors belong to DEAD/H box family of RNA-dependent ATPase, and are implicated in dynamic RNA structure rearrangement during spliceosome assembly. In mammals, however, such information is conspicuously lacking. In fact, none of the known mammalian splicing factors has characteristics for ATP hydrolysis. In an attempt to identify mammalian splicing factors involved in ATP usage, we have developed a novel approach to identify and purify spliceosomal ATP binding proteins. Six spliceosomal ATP binding proteins were found, one of them, SAFp56, was purified and microsequenced, and found to be a DEAD box protein containing unique DECD motif instead of the canonical DEAD motif. During the course of this work, a new functional region in U2AF65, an essential splicing factor required for U2 snRNP entry into the spliceosome, was defined. This information was used to clone a human U2AF65 associated protein (UAP). UAP and SAFp56 are identical. We refer to this protein as hUAP56 (human 56 kDa U2AF65 associated protein). We present evidence that hUAP56 is an essential splicing factor required for the U2 snRNP binding to pre-mRNA. Interestingly, UAP56 is recruited to pre-mRNA in a polypyrimidine tract bound U2AF65-dependent fashion. This result underscores a new function of U2AF65, and provides the first description of how a specific DEAD box protein is directed to a pre-mRNA splicing signal, and/or, to the proximity of its substrate at a particular stage. Like an authentic DEAD box protein. hUAP56 has ATP binding, RNA-stimulated ATPase, as well as RNA binding activity. A particularly novel result is that the ATPase activity of UAP56 is stimulated by U2AF65. This observation strongly suggests the role of UAP56 in ATP dependent mechanism during U2 snRNP binding to the pre-mRNA branchpoint, and implies that UAP56 may function through a distinct mechanism. We identify yeast UAP (yUAP), a highly conserved S. cerevisiae homologue of hUAP56. yUAP is essential for viability, can be functionally substituted for by hUAP56, and like its human counterpart, is an essential pre-mRNA splicing factor required for spliceosome assembly. Furthermore, we show that yUAP is required for formation of the branchpoint dependent commitment complex, the precursor for U2 snRNP addition. Site-directed mutagenesis revealed that all DEAD box protein consensus motifs are required for yUAP function. Interestingly, a strain harboring a yUAP mutant in which the DECD sequence, characteristic of UAP members, was changed to canonical sequence, is inviable. Our results demonstrate that UAP is structurally and functionally conserved from yeast to man. In conjunction with previous studies, we conclude that at least two DEAD box proteins, Prp5p and yUAP, are required for the U2 snRNP-branchpoint interaction.

Maternally inherited poly(A)+ RNAs are important for directing early development in many animal species. This thesis investigates the regulation of maternal mRNA in the South African clawed frog, Xenopus laevis. The first portion of this thesis examines an unusual class of maternal RNA, interspersed poly(A)+ RNA, which is composed of co-linear repeat and single copy sequences. A cDNA clone, called pXR, contains the repeat portion of an interspersed RNA that hybridizes to several different oocyte transcripts of diverse size that persist until the neurula stage. DNA sequence analysis of the cDNA and hybrid selection of the oocyte transcripts followed by in vitro translation show that molecules of this repeat family are not translatable. This data, combined with the developmental profile of XR containing RNAs, indicate that members of this repeat family are not likely to be maternal messenger RNAs. The second part of this thesis investigates the expression of a class of maternal mRNAs that are regulated by cytoplasmic polyadenylation during progesterone induced oocyte maturation. One particular mRNA G10, is stored as a polyadenylated RNA in the cytoplasm of stage VI oocytes until maturation when the process of poly(A) elongation stimulates its translation. Injection of mutant and wild-type mRNAs, synthesized in vitro, revealed that two sequence elements, UUUUUUAUAAAG and AAUAAA, were both necessary and sufficient for polyadenylation and polysomal recruitment of G10. Maturation promoting factor and cyclin as well as progesterone can induce polyadenylation but in each case protein synthesis is required. Extracts from oocytes and unfertilized eggs were employed to identify factors that may be responsible for maturation-specific polyadenylation. An 82 kd protein that binds to the UUUUUUAUAAAG in egg, but not oocyte extracts, was identified by UV crosslinking. This data suggests that p82 is a good candidate for a developmentally regulated protein that controls the expression of maternal messenger RNAs in early Xenopus development.

Leukocytes express a family of high molecular weight glycoproteins called leukocyte common antigens (CD45) which have tyrosine phosphatase activity and are involved in phosphotyrosine signal transduction. Antibodies to different CD45 isoforms distinguish functionally different CD4+ T cell subsets in humans, rats, and mice. Selected protein isoforms are expressed through a process of exon splicing which is cell-type and differentiation-state specific. Splicing of the three variable exons, A, B, and C, which encode amino acids located near the extracellular amino terminus of the protein, potentially results in generation of eight different mRNA transcripts. The purpose of this study was to determine the relative levels of all eight different CD45 transcripts present in a panel of murine CD4+ T cell lines and normal murine and human CD4+ T cell subsets separated with antibodies to CD45 variable exons. I show, as expected, that the broad features of CD45 surface isoform expression in these cells can be accounted for by the relative amounts of the eight differentially spliced transcripts. Unexpectedly, all the differences in CD45 isoform expression among the CD4+ T cell subpopulations that I measured could be accounted for by differences in the overall level of variable exon expression. I did not see differences among T cell populations in the relative expression of particular variable exons. Exon B was always found in greater abundance than exons C or A. Of the dual exon species, only AB and BC were found in CD4+ T cells. The AC species was undetectable. Human CD4+ T cells, especially those in the naive subset, express higher levels of CD45 variable exons than murine CD4+ T cells. In unrelated studies, I have generated a rat-mouse hybridoma which secretes a rat IgG antibody reactive with mouse CD45. I show that the monoclonal antibody, 25D10, defines a novel epitope consistent with a post-translational modification of CD45, similar but distinct from the epitope recognized by monoclonal antibody RA3.6B2 (anti-B220). This conclusion is based on evidence that it precipitates similar molecular weight bands from cells as does a framework monoclonal antibody to CD45, yet has a distinct cell surface expression as determined by flow cytometric analysis. It stains activated Th cell lines at a higher intensity than resting Th cells, stains 60-70% of splenocytes, and 25-30% of lymph node cells. It stains all class II positive cells but not freshly isolated CD4+, CD8+ T cells or CD45 transfected fibroblasts.

From the first idea, to research and comparing with other backpacks with foldable function (tele, usárna), and with backpacks associated with urban and streetwear function, I designed a backpack with a practical and functional design which opens questions of personal vs. public space. It plays with the question of lifestyle as a focal point of marketing. It speaks to young people, who spend their time in the city public space and occupy it in their own way. Technical parameters of the backpack are dependent of its functional needs, but the first idea came out of the needs of a modern young nomad, exploring the city public space.

Cleavage and polyadenylation are two necessary messenger RNA (mRNA) maturation steps for gene expression. The Cleavage and Polyadenylation Specificity Factor (CPSF) complex, which recognizes the AAUAAA polyadenylation signal and executes the cleavage reaction, is indispensable for these two processes. In this thesis, I describe my study of the regulation and functions of two non-canonical isoforms of the CPSF subunit WDR33. In addition, I provide detailed analyses on our current knowledge of CPSF subunits’ functions and their influences on a diverse collection of biological processes and conditions. In Chapter1, I provide a general introduction to cleavage and polyadenylation, WDR33, innate immune response via molecular pattern recognition, and the cGAS-STING pathway. Chapter 2 presents my original research on non-canonical WDR33 isoforms, termed WDR33v2 (V2) and WDR33v3 (V3). I determined that their mRNAs are produced by alternative polyadenylation. Both V2 and V3 proteins lack multiple WD repeats, but they can interact with and stabilize each other. This is a novel mode of protein-protein interaction, which I termed WD repeat complementation (WDRC). Unexpectedly, I found that even though V2 and V3 are isoforms of a polyadenylation factor, they are not themselves polyadenylation factors. Regulated by the NF-κB pathway, they are interestingly immune factors involved in the cGAS-STING pathway that induces immune responses against cytosolic double-stranded DNA. V2 decreases STING disulfide oligomerization and suppresses STING-mediated interferon β induction, but facilitates STING-mediated autophagy. Binding of V3 to V2 via WDRC prevents V2’s regulation of STING, suggesting that V3 is a V2 inhibitor. My findings thus further our understanding of STING-mediated immune responses. More broadly, these findings also demonstrate that isoforms produced by alternative mRNA processing can be functionally unrelated. In light of the versatility of the WDR33 gene, I performed a literature review in Chapter 3 on both the canonical and non-canonical functions of CPSF. I first summarize the general functions of CPSF subunits. Subsequently, I discuss their involvements in a variety of biological processes and conditions. This discussion reveals that different processes involve different CPSF subunits. Although CPSF is responsible for only two simple biochemical reactions, it has profound influences on cellular homeostasis. Together, my thesis studies reveal new insights into the molecular mechanism of the cGAS-STING pathway, underscore the importance of alternative mRNA processing, and provide the latest analyses of the functional significances of CPSF.

No description available.

This dissertation focuses on pre-mRNA 3'-end processing in eukaryotes, a crucial step in defining the 3'end of most protein-coding mRNAs. In vertebrates, two distinct molecular machines are involved: the canonical machinery, consisting of a Cleavage Factor (CF) module, Polyadenylation Specificity (PSF) module, Cleavage Stimulation Factor, and other complexes, and the U7 snRNP machinery (U7 machinery), which consist of a core U7 snRNP complex and the Histone Cleavage Complex (HCC). U7 snRNP is involved in replication-dependent histone pre-mRNA 3'-end processing. Interestingly, the cleavage modules of the canonical and U7 machinery share an endonuclease, CPSF73, that catalyzes the cleavage reaction for 3’-end processing of pre-mRNAs. CPSF73 also possesses 5’-3’ exonuclease activity in the U7 machinery. CPSF73 has been identified as a potential target for anticancer and antimalarial small-molecule inhibitors. Traditionally, CPSF73 nuclease activity has been demonstrated using a gel-based end-point assay, using radio-labeled or fluorescently labeled RNA substrates. In Chapter Two (Ch. 2) of this dissertation introduces a novel, real-time fluorescence assay to investigate CPSF73 nuclease activity. This efficient and high-throughput assay holds potential for identifying new CPSF73 inhibitors. Chapter Three (Ch. 3) of this dissertation delves into the structural characterization of the mammalian PSF (mPSF) module in complex with the second most frequent PAS variants, AUUAAA. Structure studies have revealed the molecular mechanism underlying mPSF recognition of the most common PAS sequence, AAUAAA. This study presents a cryo-EM structure of mPSF in complex with AUUAAA. While the binding modes remain highly similar between the two PAS variants, we observed conformational differences in the A1 and U2 nucleotides in AUUAAA compared to the A1 and A2 of AAUAAA. Furthermore, CPSF30 displayed conformational changes near the U2 nucleotide of AUUAAA. Attempts to explore the binding modes of two rare PAS sequences, AAGAAA and GAUAAA, were inconclusive due to a lack of RNA density in the EM maps. An atomic model of the ternary structure (CPSF160, WDR33, CPSF30) was produced using the EM map of the AAGAAA sample. The ternary structure revealed PAS recognizing residues to be disordered in CPSF30 (ZF2 and ZF3) and WDR33. Overall, this dissertation provides insights into the intricate mechanisms of pre-mRNA 3'-end processing in mammals, laying the groundwork for future studies and potentially leading to the development of novel inhibitors targeting CPSF73.