The splicing of U12-type introns is a rate-limiting step in gene expression

Metazoan genes have recently been found to contain a novel class of introns that display non-canonical consensus sequences and are excised by a distinct splicing machinery. These introns occur very rarely, and have been called U12-type introns because recognition of their branch point sequences requires the U12 snRNP, which performs an analogous function to the U2 snRNP in splicing major-class introns. The persistence of two spliceosomes throughout virtually all of metazoan evolution suggests that the two spliceosomes play distinct and probably indispensable cellular roles. One enticing possibility is that the U12-type spliceosome functions in post-transcriptional regulation, serving as the rate-determining step in the splicing pathway of genes containing U12-type introns. To test this idea I have investigated the timing of U12-type intron removal relative to the removal of major-class introns from pre-mRNAs that contain both intron types. I have addressed this question using two different experimental approaches.One way to evaluate the order of intron removal from a transcript is to document the relative amounts of unspliced intron sequences within a steady-state population of partially-processed transcripts: if the splicing of U12-type introns is rate limiting and occurs last, then their sequences should be more abundant than those of their major-class intron neighbors. I have developed an accurate assay based on the technique of quantitative RT-PCR to measure the relative abundance of unspliced introns within several genes. Here, I present results from the analysis of three human genes showing that in all three cases splicing of the U12-type intron proceeds more slowly than splicing of the U2-type introns from the same transcript.The second experimental approach aimed to address the question of whether replacement of a naturally occurring U12-type intron with U2-type intron consensus sequences can affect the rate of production of mature mRNA and protein. Constructs were created which expressed either cyan or yellow fluorescent proteins only when completely spliced. The constructs contained either a U12-type or a U2-type intron in an arrangement which permitted correlation of color with type of splicing. By observing the relative intensities of the two fluorescent colors, it was possible to infer the relative efficiencies of the two splicing pathways within transfected Drosophila melanogaster tissue culture cells. Results of these experiments showed that replacement of a U12-type intron with canonical consensus sequences did indeed dramatically increase expression of the corresponding mRNA and protein.These results provide direct evidence that in vivo gene expression can be altered by the presence of a U12-type intron and implicate the U12-type spliceosome as a potential target in the post-transcriptional regulation of gene expression

Diverse Forms of RPS9 Splicing Are Part of an Evolving Autoregulatory Circuit

Ribosomal proteins are essential to life. While the functions of ribosomal protein-encoding genes (RPGs) are highly conserved, the evolution of their regulatory mechanisms is remarkably dynamic. In Saccharomyces cerevisiae, RPGs are unusual in that they are commonly present as two highly similar gene copies and in that they are over-represented among intron-containing genes. To investigate the role of introns in the regulation of RPG expression, we constructed 16 S. cerevisiae strains with precise deletions of RPG introns. We found that several yeast introns function to repress rather than to increase steady-state mRNA levels. Among these, the RPS9A and RPS9B introns were required for cross-regulation of the two paralogous gene copies, which is consistent with the duplication of an autoregulatory circuit. To test for similar intron function in animals, we performed an experimental test and comparative analyses for autoregulation among distantly related animal RPS9 orthologs. Overexpression of an exogenous RpS9 copy in Drosophila melanogaster S2 cells induced alternative splicing and degradation of the endogenous copy by nonsense-mediated decay (NMD). Also, analysis of expressed sequence tag data from distantly related animals, including Homo sapiens and Ciona intestinalis, revealed diverse alternatively-spliced RPS9 isoforms predicted to elicit NMD. We propose that multiple forms of splicing regulation among RPS9 orthologs from various eukaryotes operate analogously to translational repression of the alpha operon by S4, the distant prokaryotic ortholog. Thus, RPS9 orthologs appear to have independently evolved variations on a fundamental autoregulatory circuit

Nonsense-mediated mRNA reduction and pre-mRNA processing in drosophila

From bacteria to mammalian cells, the presence of a nonsense mutation causes a reduction in the level of the mRNA of the corresponding gene. The reduction is not, contrary to initial expectations, due to a passive mechanism by which non translated mRNAs are degraded; rather it is a active process in which active translation, cis-acting sequences and specific trans-acting factors are required. It is generally accepted that this phenomenon is the consequence of an evolutionary conserved mechanism that evolved to protect cells from the potentially deleterious effect of truncated proteins - this is often referred to as the mRNA surveillance system or nonsense mediated mRNA decay (NMD). This phenomenon has been extensively studied in budding yeast and in mammalian systems and to a lesser extent in C. elegans. In yeast the recognition of the nonsense codon appears to occur during cytoplasmic translation and premature translation termination is thought to activate a specific protein complex - called the surveillance complex - which in tum triggers an accelerated decay of the aberrant mRNA. However, contrary to the expectation that the recognition of the nonsense codon should occur during cytoplasmic translation, several studies in mammalian cells indicate that NMD may take place in the nucleus by a mechanism that is independent of cytoplasmic translation. For example, several reports indicate that this reduction occurs while the mRNA is still associated with the nucleus, and that the stability of the cytoplasmic mRNA is unchanged relative to a wild-type allele. The common view in the field is that these apparently discordant results between NMD in yeast and in mammalian cells will eventually be accommodated in a single model in which translation in the cytoplasm plays a prominent role. For example, a commonly given explanation is that the recognition of the nonsense codon takes place during nuclear export, and it has been implied that the apparent effects on nuclear RNA are in fact triggered by the premature abortion of translation at the cytoplasmic side of the nuclear envelope. However not all the data from mammalian systems can be so easily explained by the above model. For example, several reports indicate that nonsense mutations affect the splicing of the corresponding pre-mRNA, which makes it difficult to imagine how premature translation in the cytoplasm could effect such an early event in mRNA biogenesis

piRNA Biogenesis and Transposon Silencing in Drosophila: A Dissertation

piRNAs guide PIWI proteins to silence transposons in animal germ cells. In Drosophila, the heterochromatic piRNA clusters transcribe piRNA precursors to be transported into nuage, a perinuclear structure for piRNA production and transposon silencing. At nuage, reciprocal cycles of piRNA-directed RNA cleavage—catalyzed by the PIWI proteins Aubergine (Aub) and Argonaute3 (Ago3) in Drosophila—destroy the sense transposon mRNA and expand the population of antisense piRNAs in response to transposon expression, a process called the Ping-Pong cycle. Heterotypic Ping-Pong between Aub and Ago3 ensures that antisense piRNAs predominate. My thesis research mainly focuses on two fundamental questions about the piRNA production: How does the germ cell differentiate piRNA precursor from mRNAs for piRNA biogenesis? And what is the mechanism to impose Aub Ping-Pong with Ago3? For the first question, we show that the HP1 homolog protein Rhino marks the piRNA cluster regions in the genome for piRNA biogenesis. Rhino seems to anchor a nuclear complex that suppresses cluster transcript splicing, which may differentiate piRNA precursors from mature mRNAs. Moreover, LacI::Rhino fusion protein binding suppresses splicing of a reporter transgene and is sufficient to trigger de novo piRNA production from a trans combination of sense and antisense transgenes. For the second question, we show that Qin, a new piRNA pathway factor contains both E3 ligase and Tudor domains, colocalizes with Aub and Ago3 in nuage, enforces heterotypic Ping- Pong between Aub and Ago3. Loss of qinleads to less Ago3 binding to Aub, futile Aub:Aub homotypic Ping-Pong prevails, antisense piRNAs decrease, many families of mobile genetic elements are reactivated, DNA damage accumulates in the germ cells and flies are sterile

A Hox gene mutation that triggers Nonsense-mediated RNA decay and affects alternative splicing during Drosophila development

Nonsense mutations are usually assumed to affect protein function by generating truncated protein products. Nonetheless, it is now clear that these mutations affect not just protein synthesis but also messenger RNA stability. The surveillance mechanism responsible for the detection and degradation of 'nonsense' RNA messages is termed nonsense-mediated RNA decay (NMD). Essential biochemical components of the NMD machinery have been defined in several species. Here we identify the Drosophila orthologue of one of these factors, Upf1, and document its expression during embryogenesis. To test whether NMD acts during Drosophila development, we make use of a mutation that introduces a stop codon into a variably spliced exon of the Hox gene Ultrabithorax (Ubx). Using real-time quantitative RT-PCR we demonstrate that Ubx transcripts containing the premature stop codon are expressed at lower levels than their wild type counterpart. Unexpectedly, we also find that the same mutation significantly increases the levels of a Ubx splicing isoform that lacks the exon containing the premature termination codon. These findings indicate that NMD is operational during Drosophila development and suggest that nonsense mutations may affect development by altering the spectrum of splicing products formed, as well as by reducing or eliminating protein synthesis

Expression of ribosomal protein L22e family members in Drosophila melanogaster: rpL22-like is differentially expressed and alternatively spliced

Several ribosomal protein families contain paralogues whose roles may be equivalent or specialized to include extra-ribosomal functions. RpL22e family members rpL22 and rpL22-like are differentially expressed in Drosophila melanogaster: rpL22-like mRNA is gonad specific whereas rpL22 is expressed ubiquitously, suggesting distinctive paralogue functions. To determine if RpL22-like has a divergent role in gonads, rpL22-like expression was analysed by qRT-PCR and western blots, respectively, showing enrichment of rpL22-like mRNA and a 34 kDa (predicted) protein in testis, but not in ovary. Immunohistochemistry of the reproductive tract corroborated testis-specific expression. RpL22-like detection in 80S/polysome fractions from males establishes a role for this tissue-specific paralogue as a ribosomal component. Unpredictably, expression profiles revealed a low abundant, alternative mRNA variant (designated ‘rpL22-like short’) that would encode a novel protein lacking the C-terminal ribosomal protein signature but retaining part of the N-terminal domain. This variant results from splicing of a retained intron (defined by non-canonical splice sites) within rpL22-like mRNA. Polysome association and detection of a low abundant 13.5 kDa (predicted) protein in testis extracts suggests variant mRNA translation. Collectively, our data show that alternative splicing of rpL22-like generates structurally distinct protein products: ribosomal component RpL22-like and a novel protein with a role distinct from RpL22-like

Function and Regulation of the Y-Linked Axonemal Dynein Genes During Drosophila Spermatogenesis

The germline is considered to be immortal, meaning an organism’s germ cells have the potential to give rise to all subsequent generations. With so much at stake, the germline goes to great lengths to protect itself while also maintaining reproductive potential, resulting in fascinating and innovative biology. This thesis focuses on two aspects of germ cell development in Drosophila males that appear disadvantageous yet are prevalent across drosophilids. The first is the expression of the Y chromosome gigantic genes – these genes are essential for fertility yet they are riddled with megabases of repetitive DNA. The second is the assembly of the long cilia found within the sperm’s tail – Drosophila have some of the longest sperm in the animal kingdom, yet little is known about how these long cilia are assembled. This thesis will describe the innovations that have allowed germ cells to overcome these challenges and will go on to discuss how these burdens may benefit the fly. In Drosophila, the Y chromosome is largely heterochromatic, encoding only a handful of genes, which are essential for male fertility. Intriguingly, some of these genes are amongst the largest genes identified to date, spanning several megabases. For example, the gene kl-3, which encodes an axonemal dynein motor protein required for sperm motility, spans 4.3Mb with only 14kb of coding sequence. The introns of these genes contain megabases of simple satellite DNA repeats (e.g. (AATAT)n) that comprise over 99% of the locus. Although this “intron gigantism” has been observed in several genes across species, including the mammalian Dystrophin gene, its regulation and functional relevance remains elusive. The transcription/processing of such gigantic genes/RNA transcripts poses a significant challenge. I identified that the Y-linked gigantic genes require a unique gene expression program in order to overcome these challenges. By monitoring Y-linked gene expression over developmental time, I found that transcription of these loci takes 80-90 hours. I further identified two RNA-binding proteins that specifically bind to Y-linked gene transcripts. Loss of either RNA binding protein resulted in sterility due to the loss of Y-linked gene products. I found that this unique gene expression program functions on two fronts: it increases the ability of RNA polymerase to transcribe the repetitive introns, and it aids in processing the large transcripts. I speculate that this program may be utilized to modulate gene expression patterns during development. During Drosophila spermatogenesis, germ cells undergo drastic morphological changes to yield a 1.9mm sperm. The cilia found within the sperm tail are cytoplasmic cilia – a specialized type of cilia where the axoneme (the microtubule structural component) resides within the cytoplasm instead of within a specialized ciliary compartment. Cytoplasmic cilia likely allow for efficient assembly of longer cilia, however, the mechanism for their assembly remains unknown. I found that mRNAs encoding four axonemal dynein heavy chain genes (three of which are Y-linked gigantic genes) colocalize in a novel ribonucleoprotein (RNP) granule, which localizes near the site of axoneme assembly during sperm elongation. Precise localization of this RNP granule mediates incorporation of the axonemal dynein motor proteins into the axoneme. This work is the first to uncover how cytoplasmic cilia are efficiently assembled to allow for the production of 1.9mm sperm, and highlights that there are other cilia assembly mechanisms besides the ancient and conserved mechanism by which traditional cilia assemble.PHDCellular & Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/163043/1/jaclynmf_1.pd