Regulating transcript fate through RNA elements in the 3' untranslated region
AuthorIdler, Randy Keegan
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Regulation of protein production is the most vital part of molecular biology. Typically the focus of such endeavors has been on the regulation of transcription, but increasingly there has been a concentration on the post-transcriptional regulation of mRNAs via miRNAs, other small RNAs, and RNA binding proteins. Controlled translation of transcripts has been shown to play a vital role in rapid response to changing conditions such as dendritic tree formation, ischemia, heat shock, and immune response. Such regulation also performs a vital role in development of embryos, oocytes, and spermatozoa. Spermatogenesis is the process of the creation of spermatozoa from primordial germ cells. For mice, this lengthy process begins in the womb while the fetus is still developing, and the first functional spermatozoa are produced more than a month after birth. Spermiogenesis is the haploid stages of spermatogenesis; the first wave of this progress from round spermatids to elongated spermatids occurs roughly from post-natal day 21 to post-natal day 35. Tightly regulated temporal control of translation is essential to proper sperm development. During spermiogenesis, chromatin becomes tightly wrapped around transition proteins and finally protamine, resulting in highly condensed chromatin. Once this occurs, transcription is effectively terminated, yet development continues for more than a week as elongating spermatids develop into functional spermatozoa. The transcripts for late stage proteins are kept in storage by two main mechanisms: separation from translational machinery by compartmentalization in ribonuclear protein particles, and physically hindering the binding of translational machinery by allosteric inhibition and steric hindrance. A well studied example of a protein that regulates translation by sequestering transcripts away from translational machinery is ELAVL1/HuR. In male germ cells, this protein binds transcripts in the nucleus and regulates their transport into male germ cell specific ribonucleic protein particles (RNP), known as chromatoid bodies, for storage from spermatocytes until mid-spermiogenesis. At step V of elongation, ELAVL1 moves from chromatoid bodies to polysomes, facilitating translation. CPEB is a family of well studied proteins that takes advantage of the second type of mechanism of post-transcriptional regulation whereby it prevents the binding of translational machinery. It does this primarily by controlling the length of the poly(A) tail, which is then bound by PABP leading to a cascade of interactions resulting in the recruitment of translational machinery. This mechanism is more nuanced than isolating transcripts away from translational machinery, as the length of the poly (A) tail determines how many PABP molecules attach and consequently the proportion of transcripts that undergo translation. Both sequestering of transcripts and inhibition of binding of translational machinery are controlled in large part by RNA binding proteins, which recognize specific RNA structures and sequences located primarily in the 3' untranslated region (3'UTR), termed RNA motifs or RNA elements. A common mechanism of post-transcriptional control is guided by small non-coding RNAs (sncRNAs) such as microRNA or piRNA, where proteins are guided to their target transcripts via complementary binding of their associated sncRNAs. Competition for binding sites by miRNA guided proteins and element recognition proteins represents an intriguing new field. Provided in this analysis is an in depth discussion of known RBPs, RNPs, sncRNAs, elements and regulatory mechanisms showing the current state of the literature. Norman Hecht is a leader in the field of molecular mechanisms of transcript fate control in spermatogenesis. His lab previously performed a study detailing transcript association with both messenger ribonuclearprotein (mRNP) and with polysomes in testis tissue at three critical time points, postnatal days 17, 21 and adult. A relative increase in association with polysomes was presumed to be indicative of increased translation and a comparison with the literature validated this assumption. Postnatal day 17 corresponds to the emergence of pachytene spermatocytes; postnatal day 21 corresponds to the emergence of round spermatids; and adult testis tissue can be assumed to correspond with the emergence of elongating spermatids, since mature spermatozoa have stopped translation. Hecht's study detailed which transcripts exist at each time point and their association with mRNP and polysomes, splitting them into groups based on their presence and movement between mRNP and polysomes at each time period. This resulted in six groups of transcripts expressed during each time period that were of particular importance for the focus of this thesis: transcripts that show increased/decreased association with polysomes from day 17 to day 21, from day 17 to adult, and from day 21 to adult. An additional group of genes showed increased association with polysomes from day 21 to adult, but was absent from the testis during day 17. Transcripts that existed over all three time points that showed no change in association between polysomes and mRNP provided a useful background against which comparisons were possible. A list of miRNAs that can potentially bind to the transcripts of each group was established using MicroCosm, an miRNA matching program with an internal database of known miRNAs and 3'UTRs transcripts. A comparison of the miRNAs corresponding to each group was inconclusive. While there were several miRNAs that showed overrepresentation in the translationally downregulated groups, only a single miRNA showed statistical significance; and while there were several miRNAs that showed underrepresentation in the translationally upregulated groups, none were statistically significant. Using Sequery, a novel in house program designed by Grant Hennig for comparison of sequences within each group, the 3'UTRs of each group were compared against the unregulated group. A biased search using motifs known to be involved in spermatogenesis was conducted, and a comparison between different groups was performed using these results. Several of these motifs were shown to be significantly overrepresented in the group showing increased translation between day 17 and adult. Interestingly, RNA motifs recognized by ELAVL1 and CPEB were among those that were overrepresented in this group and were also significantly underrepresented in the group transcribed beginning day 21 and showing upregulation of translation from day 21 to adult. An unbiased search of various lengths from 7nt to 20nt was also performed using an exact matching protocol and using a matching protocol that allows substitutions. A comparison between groups revealed many potential elements that were significantly overrepresented in the groups that showed regulated translation over the three time points, especially in the group that showed upregulation from day 17 to adult. In order to hone the potential elements and increase the likelihood that they are of biological importance, a qualitative procedure was performed that looked for similarities between potential elements. Though the results of this study were promising, further study is needed to validate these presumptive motifs.