Details

Autor(en) / Beteiligte

• Rodríguez Martínez, Deyra Nichole

Titel

Characterization of Lin28-RNA Interactions in Human Embryonic Stem Cells

Ort / Verlag

ProQuest Dissertations & Theses

Erscheinungsjahr

2014

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• RNA binding proteins (RBPs) play essential roles in the post-transcriptional regulation of gene expression. The RBP Lin28 is highly expressed in human embryonic stem (hES) cells and is known to play important roles in hES cell self-renewal and pluripotency. However, it is not completely understood how Lin28 contributes to the regulation of stem cell properties. This dissertation aims to advance our understanding on the molecular mechanisms Lin28 utilizes to influence stem cell biology. The first goal of the study was to systematically identify the full complement of Lin28-associated RNAs in hES cells. Using crosslinking and immunoprecipitation (CLIP) in combination with other genome-wide in vivo and in vitro techniques, we discovered that Lin28 interacts with the large majority of cellular RNAs, including all major functional classes of cytosolic RNAs in hES cells. Among the large number of targets preferential association occurs with tRNAs and mRNAs. The high abundance of Lin28 in hES cells is sufficient to allow binding of multiple Lin28 molecules to most of its mRNA targets. The second goal of the study was to uncover Lin28 recognition sequences on the identified RNA targets. Bioinformatic analysis and in vitro testing of Lin28 binding regions showed that Lin28 recognition of RNA targets occurs through interactions with low complexity nucleotide motifs that vary among RNA classes. The motif preference was clearest in mRNAs where Lin28 bound preferentially but not exclusively to the 3'UTR and favored interactions with AU rich sequences. The third and final goal of the study was to better understand Lin28's role in regulation of its mRNA targets. The data showed that in hES cells Lin28 appears to play no more than a minor role in regulating mRNA stability. However, Lin28 binding is strongly associated with a Lin28-dependent decrease in the density of ribosomes associated with its mRNA targets. The multiplicity of Lin28's interactions with diverse functional classes of RNA in hES cells, and the evidence that these interactions systematically alter translation of its mRNA targets, suggest that Lin28 may have a general transcriptome-wide regulatory role in hES cells.