Messenger RNA half-life measurements in mammalian cells.

The recognition of the importance of mRNA turnover in regulating eukaryotic gene expression has mandated the development of reliable, rigorous, and "user-friendly" methods to accurately measure changes in mRNA stability in mammalian cells. Frequently, mRNA stability is studied indirectly by analyzing the steady-state level of mRNA in the cytoplasm; in this case, changes in mRNA abundance are assumed to reflect only mRNA degradation, an assumption that is not always correct. Although direct measurements of mRNA decay rate can be performed with kinetic labeling techniques and transcriptional inhibitors, these techniques often introduce significant changes in cell physiology. Furthermore, many critical mechanistic issues as to deadenylation kinetics, decay intermediates, and precursor-product relationships cannot be readily addressed by these methods. In light of these concerns, we have previously reported transcriptional pulsing methods based on the c-fos serum-inducible promoter and the tetracycline-regulated (Tet-off) promoter systems to better explain mechanisms of mRNA turnover in mammalian cells. In this chapter, we describe and discuss in detail different protocols that use these two transcriptional pulsing methods. The information described here also provides guidelines to help develop optimal protocols for studying mammalian mRNA turnover in different cell types under a wide range of physiologic conditions.

A cellular cleavage pathway for the Moloney murine leukemia virus precursor protein, Pr65$\rm\sp{gag}$: Identification of a new viral protein containing CAp30 and NCp10 sequences

The origin and structure of P55$\sp{\rm gag},$ a gag encoded polyprotein lacking the nucleocapsid protein, NCp10, have been explored. Evidence shows that P55$\sp{\rm gag}$ is formed by non-viral proteolytic cleavage of the Moloney murine leukemia virus (MoMuLV)gag precursor protein, Pr65$\sp{\rm gag}.$ P55$\sp{\rm gag}$ is produced in cells infected by a viral protease deletion mutant and by a recombinant murine sarcoma virus known to lack the protease gene, implying that a cellular protease is responsible for the cleavage. Structural and immunological studies show that the protein cleavage site is upstream of the CAp30-NCp10 viral proteolytic junction, implying that P55$\sp{\rm gag}$ lacks the carboxy-terminal residues of CAp30. During the course of studying P55$\sp{\rm gag},$ another protein was discovered, which I named nucleocapsid-related protein(NCRP). NCRP possesses the portion of CAp30 that is lacking in P55$\sp{\rm gag}.$ NCRP possesses antigenic epitopes present in CAp30 and NCp10. NCRP was observed in virus lysates and in nuclear lysates of MoMuLV infected cells; it was not detected in the cytoplasmic fractions of MoMuLV infected cells. Our results indicated that NCRP originates from Pr65$\sp{\rm gag},$ resulting from the same cellular proteolytic cleavage event that produces the viral cellular protein P55$\sp{\rm gag}.$ P55$\sp{\rm gag}$- and NCRP-like proteins also were observed in AKV murine leukemia virus (AKV MuLV) and feline leukemia virus (FeLV) infected cells and in their respective virus particles. The site of cleavage that yields P55$\sp{\rm gag}$ and NCRP is within the carboxy terminus of CAp30, likely within a motif highly conserved among mammalian type C retroviruses. This new motif, called the capsid conserved motif (CCM), overlaps a region containing both a possible bipartite nuclear targeting sequence and a region homologous with the U1 small nuclear ribonucleoprotein 70-kD protein. This domain, when intact, may act as a nuclear targeting sequence for the gag precursor proteins Pr65$\sp{\rm gag}$ and CAp30. Nuclei of cells infected with MoMuLV were examined for the presence of gag proteins. Both Pr65$\sp{\rm gag}$ and CAp30 were detected in the nuclear fraction of MoMuLV, AKV MuLV and FeLV infected cells. P55$\sp{\rm gag}$ was never detected in the nucleus of MoMuLV, AKV MuLV and FeLV infected cells or in their respective virus particles. I propose that NCRP may be involved in sequestering viral genomic RNA for the purposes of encapsidation and intracellular viral genomic RNA dimerization. ^

Correction of aberrant FGFR1 RNA splicing through blocking intronic regulatory elements

Analysis of the human genome has revealed that more than 74% of human genes undergo alternative RNA splicing. Aberrations in alternative RNA splicing have been associated with several human disorders, including cancer. ^ We studied the aberrant expression of alternative RNA splicing isoforms of the Fibroblast Growth Factor Receptor 1 (FGFR1) gene in a human glioblastoma cancer model. Normal glial cells express the FGFR1α, which contains three extracellular domains. In tumors the most abundant isoform is the FGFR1β, which lacks the first extracellular domain due to the skipping of a single exon, termed alpha. The skipping of the α-exon is regulated by two intronic silencing sequences within the precursor mRNA. Since we observed no mutations on these elements in tumor cells, we hypothesized that the over-expression of regulatory proteins that recognize these sequences is responsible for the aberrant expression of splicing isoforms. Hence, we blocked the formation of protein complexes on the ISS using antisense RNA oligonucleotides in vitro. We also evaluated the impact of the ISS antisense oligonucleotides on the endogenous FGFR1 splicing, in a glioblastoma cell model. By targeting intronic regulatory elements we were able to increase the level of alpha exon inclusion up to 90% in glioblastoma cells. The effect was dose dependent, sequence specific and reproducible in glioblastoma and other cancer cells, which also exhibit an alpha exon skipping phenotype. Targeting FGFR1 endogenous ISS1 and ISS2 sequences did not have an additive or synergistic effect, which suggest a regulatory splicing mechanism that requires the interaction of complexes formed on these elements. An increase in the levels of the FGFR1α isoform resulted in a reduction in cell invasiveness. Also, a significant increase in the levels of caspase 3/7 activities, which is indicative of an elevation in apoptosis levels, suggests that expression of FGFR1β might be relevant for tumor survival. These studies demonstrate that it is possible to prevent aberrant expression of exon skipping events through the targeting of intronic regulatory elements, providing an important new therapeutic tool for the correction of human disease caused by alternative RNA splicing. ^

Regulation of RNA splicing by nonsense mutations

Translation termination as a result of premature nonsense codon-incorporation in a RNA transcript can lead to the production of aberrant proteins with gain-of-function or dominant negative properties that could have deletrious effects on the cell. T-cell Receptor (TCR) genes acquire premature termination codons two-thirds of the time as a result of the error-prone programmed rearrangement events that normally occur during T-cell development. My studies have focused on the fate of TCR precursor mRNAs in response to in-frame nonsense mutations. ^ Previous published studies from our laboratory have shown that TCR precursor mRNAs are subject to nonsense mediated upregulation of pre-mRNA (NMUP). In this dissertation, I performed substitution and deletion analysis to characterize specific regions of TCR which are required to elicit NMUP. I performed frame- and factor-dependence studies to determine its relationship with other nonsense codon induced responses using several approaches including (i) translation dependence studies (ii) deletion and mutational analysis, as well as (iii) siRNA mediated knockdown of proteins involved. I also addressed the underlying molecular mechanism for this pre-mRNA upregulation by (i) RNA half-life studies using a c-fos inducible promoter, and (ii) a variety of assays to determine pre-mRNA splicing efficiency. ^ Using these approaches, I have identified a region of TCR that is both necessary and sufficient to elicit (NMUP). I have also found that neither cytoplasmic translation machinery nor the protein UPF1 are involved in eliciting this nuclear event. I have shown that the NMUP can be induced not only by nonsense and frameshift mutations, but also missense mutations that disrupt a cis splicing element in the exon that contains the mutation. However, the effect of nonsense mutations on pre-mRNA is unique and distinguishable from that of missense mutations in that nonsense mutations can upregulate pre-mRNA in a frame-dependent manner. Lastly, I provide evidence that NMUP occurs by a mechanism in which nonsense mutations inhibit the splicing of introns. In summary, I have found that TCR precursor mRNAs are subject to multiple forces involving both RNA splicing and translation that can either increase or decrease the levels of these precursor mRNAs. ^

A NEW TUMOR SUPPRESSOR GENE CANDIDATE REGULATED BY THE NON-CODING RNA PCA3 IN HUMAN PROSTATE CANCER

Prostate cancer is the second leading cause of cancer-related death and the most common non-skin cancer in men in the USA. Considerable advancements in the practice of medicine have allowed a significant improvement in the diagnosis and treatment of this disease and, in recent years, both incidence and mortality rates have been slightly declining. However, it is still estimated that 1 man in 6 will be diagnosed with prostate cancer during his lifetime, and 1 man in 35 will die of the disease. In order to identify novel strategies and effective therapeutic approaches in the fight against prostate cancer, it is imperative to improve our understanding of its complex biology since many aspects of prostate cancer initiation and progression still remain elusive. The study of tumor biomarkers, due to their specific altered expression in tumor versus normal tissue, is a valid tool for elucidating key aspects of cancer biology, and may provide important insights into the molecular mechanisms underlining the tumorigenesis process of prostate cancer. PCA3, is considered the most specific prostate cancer biomarker, however its biological role, until now, remained unknown. PCA3 is a long non-coding RNA (ncRNA) expressed from chromosome 9q21 and its study led us to the discovery of a novel human gene, PC-TSGC, transcribed from the opposite strand and in an antisense orientation to PCA3. With the work presented in this thesis, we demonstrate that PCA3 exerts a negative regulatory role over PC-TSGC, and we propose PC-TSGC to be a new tumor suppressor gene that contrasts the transformation of prostate cells by inhibiting Rho-GTPases signaling pathways. Our findings provide a biological role for PCA3 in prostate cancer and suggest a new mechanism of tumor suppressor gene inactivation mediated by non-coding RNA. Also, the characterization of PCA3 and PC-TSGC led us to propose a new molecular pathway involving both genes in the transformation process of the prostate, thus providing a new piece of the jigsaw puzzle representing the complex biology of prostate cancer.

THE DOMAINS OF THE CATALYTIC SUBUNIT OF THE EUKARYOTIC RNA DEGRADING EXOSOME, RRP44P, HAVE DISTINCT FUNCTIONS

The exosome is a 3’ to 5’ exoribonuclease complex that consists of ten essential subunits. In the cytoplasm, the exosome degrades mRNA in a general mRNA turnover pathway and in several mRNA surveillance pathways. In the nucleus, the exosome processes RNA precursors to form small, stable, mature RNA species, including rRNA, snRNA, and snoRNA. In addition to processing these RNAs, the nuclear exosome is also involved in degrading aberrantly processed forms of these RNAs, and others, including mRNA. The 3’ to 5’ exoribonuclease activity of the exosome is contributed by the RNB domain of the only catalytically active subunit, Rrp44p, a member of the RNase II family of enzymes. In addition to the RNB domain, Rrp44p consists of three putative RNA binding domains and has an uncharacterized N-terminus, which includes a CR3 region and PIN domain. In an effort to characterize the cellular functions of the domains of Rrp44p, this study identified a second nuclease active site in the PIN domain. Specifically, the PIN domain exhibits endoribonuclease activity in vitro and is essential for exosome function. Further analysis of the nuclease activities of Rrp44p indicate a role for the exoribonuclease activity of Rrp44p in the cytoplasmic and nuclear exosome. This work has also characterized the CR3 region of Rrp44p, a region that has not yet been characterized in any other protein. This region is needed for the majority, if not all, of the cytoplasmic exosome functions as well as for interaction with the exosome. The CR3 region, along with a histidine residue in the N-terminus of Rrp44p, may coordinate a zinc atom. Preliminary evidence supports a role for this coordination in exosome function. Further investigation, however, is needed to determine the molecular dependence of the exosome on the CR3 region of Rrp44p. Despite its initial discovery thirteen years ago, the essential function of Rrp44p, and the exosome, is not yet known. The studies presented here, however, indicate that the essential function of Rrp44p and the exosome is in the nucleus and depends on the nuclease activities of Rrp44p.

Single-stranded DNA-binding proteins regulate the abundance of LIM domain and LIM domain-binding proteins.

The LIM domain-binding protein Ldb1 is an essential cofactor of LIM-homeodomain (LIM-HD) and LIM-only (LMO) proteins in development. The stoichiometry of Ldb1, LIM-HD, and LMO proteins is tightly controlled in the cell and is likely a critical determinant of their biological actions. Single-stranded DNA-binding proteins (SSBPs) were recently shown to interact with Ldb1 and are also important in developmental programs. We establish here that two mammalian SSBPs, SSBP2 and SSBP3, contribute to an erythroid DNA-binding complex that contains the transcription factors Tal1 and GATA-1, the LIM domain protein Lmo2, and Ldb1 and binds a bipartite E-box-GATA DNA sequence motif. In addition, SSBP2 was found to augment transcription of the Protein 4.2 (P4.2) gene, a direct target of the E-box-GATA-binding complex, in an Ldb1-dependent manner and to increase endogenous Ldb1 and Lmo2 protein levels, E-box-GATA DNA-binding activity, and P4.2 and beta-globin expression in erythroid progenitors. Finally, SSBP2 was demonstrated to inhibit Ldb1 and Lmo2 interaction with the E3 ubiquitin ligase RLIM, prevent RLIM-mediated Ldb1 ubiquitination, and protect Ldb1 and Lmo2 from proteasomal degradation. These results define a novel biochemical function for SSBPs in regulating the abundance of LIM domain and LIM domain-binding proteins.

Repeat length and RNA expression level are not primary determinants in CUG expansion toxicity in Drosophila models.

Evidence for an RNA gain-of-function toxicity has now been provided for an increasing number of human pathologies. Myotonic dystrophies (DM) belong to a class of RNA-dominant diseases that result from RNA repeat expansion toxicity. Specifically, DM of type 1 (DM1), is caused by an expansion of CUG repeats in the 3'UTR of the DMPK protein kinase mRNA, while DM of type 2 (DM2) is linked to an expansion of CCUG repeats in an intron of the ZNF9 transcript (ZNF9 encodes a zinc finger protein). In both pathologies the mutant RNA forms nuclear foci. The mechanisms that underlie the RNA pathogenicity seem to be rather complex and not yet completely understood. Here, we describe Drosophila models that might help unravelling the molecular mechanisms of DM1-associated CUG expansion toxicity. We generated transgenic flies that express inducible repeats of different type (CUG or CAG) and length (16, 240, 480 repeats) and then analyzed transgene localization, RNA expression and toxicity as assessed by induced lethality and eye neurodegeneration. The only line that expressed a toxic RNA has a (CTG)(240) insertion. Moreover our analysis shows that its level of expression cannot account for its toxicity. In this line, (CTG)(240.4), the expansion inserted in the first intron of CG9650, a zinc finger protein encoding gene. Interestingly, CG9650 and (CUG)(240.4) expansion RNAs were found in the same nuclear foci. In conclusion, we suggest that the insertion context is the primary determinant for expansion toxicity in Drosophila models. This finding should contribute to the still open debate on the role of the expansions per se in Drosophila and in human pathogenesis of RNA-dominant diseases.

Rtr1 is the Saccharomyces cerevisiae homolog of a novel family of RNA polymerase II-binding proteins.

Cells must rapidly sense and respond to a wide variety of potentially cytotoxic external stressors to survive in a constantly changing environment. In a search for novel genes required for stress tolerance in Saccharomyces cerevisiae, we identified the uncharacterized open reading frame YER139C as a gene required for growth at 37 degrees C in the presence of the heat shock mimetic formamide. YER139C encodes the closest yeast homolog of the human RPAP2 protein, recently identified as a novel RNA polymerase II (RNAPII)-associated factor. Multiple lines of evidence support a role for this gene family in transcription, prompting us to rename YER139C RTR1 (regulator of transcription). The core RNAPII subunits RPB5, RPB7, and RPB9 were isolated as potent high-copy-number suppressors of the rtr1Delta temperature-sensitive growth phenotype, and deletion of the nonessential subunits RPB4 and RPB9 hypersensitized cells to RTR1 overexpression. Disruption of RTR1 resulted in mycophenolic acid sensitivity and synthetic genetic interactions with a number of genes involved in multiple phases of transcription. Consistently, rtr1Delta cells are defective in inducible transcription from the GAL1 promoter. Rtr1 constitutively shuttles between the cytoplasm and nucleus, where it physically associates with an active RNAPII transcriptional complex. Taken together, our data reveal a role for members of the RTR1/RPAP2 family as regulators of core RNAPII function.

Astrocytes from the contused spinal cord inhibit oligodendrocyte differentiation of adult oligodendrocyte precursor cells by increasing the expression of bone morphogenetic proteins.

Promotion of remyelination is an important therapeutic strategy to facilitate functional recovery after traumatic spinal cord injury (SCI). Transplantation of neural stem cells (NSCs) or oligodendrocyte precursor cells (OPCs) has been used to enhance remyelination after SCI. However, the microenvironment in the injured spinal cord is inhibitory for oligodendrocyte (OL) differentiation of NSCs or OPCs. Identifying the signaling pathways that inhibit OL differentiation in the injured spinal cord could lead to new therapeutic strategies to enhance remyelination and functional recovery after SCI. In the present study, we show that reactive astrocytes from the injured rat spinal cord or their conditioned media inhibit OL differentiation of adult OPCs with concurrent promotion of astrocyte differentiation. The expression of bone morphogenetic proteins (BMP) is dramatically increased in the reactive astrocytes and their conditioned media. Importantly, blocking BMP activity by BMP receptor antagonist, noggin, reverse the effects of active astrocytes on OPC differentiation by increasing the differentiation of OL from OPCs while decreasing the generation of astrocytes. These data indicate that the upregulated bone morphogenetic proteins in the reactive astrocytes are major factors to inhibit OL differentiation of OPCs and to promote its astrocyte differentiation. These data suggest that manipulation of BMP signaling in the endogenous or grafted NSCs or OPCs may be a useful therapeutic strategy to increase their OL differentiation and remyelination and enhance functional recovery after SCI.

Coordinated changes in mRNA turnover, translation, and RNA processing bodies in bronchial epithelial cells following inflammatory stimulation.

Bronchial epithelial cells play a pivotal role in airway inflammation, but little is known about posttranscriptional regulation of mediator gene expression during the inflammatory response in these cells. Here, we show that activation of human bronchial epithelial BEAS-2B cells by proinflammatory cytokines interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-alpha) leads to an increase in the mRNA stability of the key chemokines monocyte chemotactic protein 1 and IL-8, an elevation of the global translation rate, an increase in the levels of several proteins critical for translation, and a reduction of microRNA-mediated translational repression. Moreover, using the BEAS-2B cell system and a mouse model, we found that RNA processing bodies (P bodies), cytoplasmic domains linked to storage and/or degradation of translationally silenced mRNAs, are significantly reduced in activated bronchial epithelial cells, suggesting a physiological role for P bodies in airway inflammation. Our study reveals an orchestrated change among posttranscriptional mechanisms, which help sustain high levels of inflammatory mediator production in bronchial epithelium during the pathogenesis of inflammatory airway diseases.

Wingless activity in the precursor cells specifies neuronal migratory behavior in the Drosophila nerve cord.

Neurons and their precursor cells are formed in different regions within the developing CNS, but they migrate and occupy very specific sites in the mature CNS. The ultimate position of neurons is crucial for establishing proper synaptic connectivity in the brain. In Drosophila, despite its extensive use as a model system to study neurogenesis, we know almost nothing about neuronal migration or its regulation. In this paper, I show that one of the most studied neuronal pairs in the Drosophila nerve cord, RP2/sib, has a complicated migratory route. Based on my studies on Wingless (Wg) signaling, I report that the neuronal migratory pattern is determined at the precursor cell stage level. The results show that Wg activity in the precursor neuroectodermal and neuroblast levels specify neuronal migratory pattern two divisions later, thus, well ahead of the actual migratory event. Moreover, at least two downstream genes, Cut and Zfh1, are involved in this process but their role is at the downstream neuronal level. The functional importance of normal neuronal migration and the requirement of Wg signaling for the process are indicated by the finding that mislocated RP2 neurons in embryos mutant for Wg-signaling fail to properly send out their axon projection.

A molecular analysis of the conditional defect in MuSVts 110 viral RNA splicing

Cells infected with MuSVts110 express a viral RNA which contains an inherent conditional defect in RNA splicing. It has been shown previously that splicing of the MuSVts110 primary transcript is essential to morphological transformation of 6m2 cells in vitro. A growth temperature of 33$\sp\circ$C is permissive for viral RNA splicing,and, consequently, 6m2 cells appear morphologically transformed at this temperature. However, 6m2 cells appear phenotypically normal when incubated at 39$\sp\circ$C, the non-permissive temperature for viral RNA splicing.^ After a shift from 39$\sp\circ$C to 33$\sp\circ$C, the coordinate splicing of previously synthesized and newly transcribed MuSVts110 RNA was achieved. By S1 nuclease analysis of total RNA isolated at various times, 5$\sp\prime$ splice site cleavage of the MuSVts110 transcript appeared to occur 60 minutes after the shift to 33$\sp\circ$C, and 30 minutes prior to detectable exon ligation. In addition, consistent with the permissive temperatures and the kinetic timeframe of viral RNA splicing after a shift to 33$\sp\circ$C, four temperature sensitive blockades to primer extension were identified 26-75 bases upstream of the 3$\sp\prime$ splice site. These blockades likely reflect four branchpoint sequences utilized in the formation of MuSVts110 lariat splicing-intermediates.^ The 54-5A4 cell line is a spontaneous revertant of 6m2 cells and appears transformed at all growth temperatures. Primer extension sequence analysis has shown that a five base deletion occurred at the 3$\sp\prime$ splice site in MuSVts110 RNA allowing the expression of a viral transforming protein in 54-5A4 in the absence of RNA splicing, whereas in the parental 6m2 cell line, a splicing event is necessary to generate a similar transforming protein. As a consequence of this deletion, splicing cannot occur and the formation of the four MuSVts110 branched-intermediates were not observed at any temperature in 54-5A4 cells. However, 5$\sp\prime$ splice site cleavage was still detected at 33$\sp\circ$C.^ Finally, we have investigated the role of the 1488 bp deletion which occurred in the generation of MuSVts110 in the activation of temperature sensitive viral RNA splicing. This deletion appears solely responsible for splice site activation. Whether intron size is the crucial factor in MuSVts110 RNA splicing or whether inhibitory sequences were removed by the deletion is currently unknown. (Abstract shortened with permission of author.) ^

Ca++/calmodulin-dependent protein kinase II: Messenger RNA in developing rat brain

Ca$\sp{++}$/calmodulin-dependent protein kinase II (CaM-KII) is highly concentrated in mammalian brain, comprising as much as 2% of the total protein in some regions. In forebrain, CaM-KII has been shown to be enriched in postsynaptic structures where it has been implicated in maintaining cytoskeletal structure, and more recently in signal transduction mechanisms and processes underlying learning and memory. CaM-KII appears to exist as a holoenzyme composed of two related yet distinct subunits, alpha and beta. The ratio of the subunits in the holoenzyme varies with different brain regions and to some degree with subcellular fractions. The two subunits also display distinct developmental profiles. Levels of alpha subunit are not evident at birth but increase dramatically during postnatal development, while levels of beta subunit are readily detected at birth and only gradual increase postnatally. The distinct regional, subcellular and developmental distribution of the two subunits of CaM-KII have prompted us to examine factors involved in regulating the synthesis of the subunit proteins.^ This dissertation addresses the regional and developmental expression of the mRNAs for the individual subunits using in situ hybridization histochemistry and northern slot-blot analysis. By comparing the developmental profile of each mRNA with that of its respective protein, we have determined that initiation of gene transcription is likely the primary site for regulating CaM-KII protein levels. Furthermore, the distinct cytoarchitecture of the hippocampus has allowed us to demonstrate that the alpha, but not beta subunit mRNA is localized in dendrites of certain forebrain neurons. The localization of alpha subunit mRNA at postsynaptic structures, in concert with the accumulation of subunit protein, suggests that dendritic synthesis of CaM-KII alpha subunit may be important for maintaining postsynaptic structure and/or function. ^

Exon involvement in the regulation of MuSVts110 RNA splicing

MuSVts110 is a conditionally defective mutant of Moloney murine sarcoma virus which undergoes a novel tmperature-dependent splice event at growth temperatures of 33$\sp\circ$C or lower. Relative to wild-type MuSV-124, MuSVts110 contains a 1487 base deletion spanning from the 3$\sp\prime$ end of the p30 gag coding region to just downstream of the first v-mos initiation codon. As a result, the gag and mos genes are fused out of frame and no v-mos protein is expressed. However, upon a shift to 33$\sp\circ$C or lower, a splice event occurs which removes 431 bases, realigns the gag and mos genes, and allows read-through translation of a P85gag-mos transforming protein. Interestingly, while the cryptic splice sites utilized in MuSVts110 are present and unaltered in MuSV-124, they are never used. Due to the 1487 base deletion, the MuSV-124 intron was reduced from 1919 to 431 bases suggesting that intron size might be involved in the activation of these cryptic splice sites in MuSVts110. Since the splicing phenotype of the MuSVts110 equivalent (TS32 DNA) which contains the identical 1487 base deletion introduced into otherwise wild-type MuSV-124 DNA, was indistinguishable from authentic MuSVts110, it was concluded that this deletion alone is responsible for activation of the cryptic splice sites used in MuSVts110. These results also confirmed that thermodependent splicing is an intrinsic property of the viral RNA and not due to some cellular defect. Furthermore, analysis of gag gene deletion and frameshift MuSVts110 mutants demonstrated that viral gag gene proteins do not play a role in regulation of MuSVts110 splicing. Instead, cis-acting viral sequences appear to mediate regulation of the splice event.^ Our initial observation that truncation of the MuSVts110 transcript, leaving only residual amounts of the flanking exon sequences, completely abolished splicing activity argued that exon sequences might participate in the regulation of the splice event.^ Analysis of exon sequence involvement has also identified cis-acting sequences important in the thermodependence of the splice event. Data suggest that regulation of the MuSVts110 splice event involves multiple interactions between specific intron and exon sequences and spliceosome components which together limit splicing activity to temperatures of 33$\sp\circ$C or lower while simultaneously restricting splicing to a maximum of 50% efficiency. (Abstract shortened with permission of author.) ^