This study integrated phylogeny, expression, and targeting information to understand the functional and evolutionary roles of miRNAs, with a focus on human miRNAs. The study classified miRNAs by conservation levels and found that more conserved miRNAs are usually expressed more abundantly in more tissues, target more genes, and are less likely to change in sequence than less conserved miRNAs. It also found that more conserved miRNAs have stronger control over the expression of their target genes. The study proposed two models of miRNA involvement in tissue complexity and found that miRNAs likely have important roles in establishing new tissue identities but may be replaced by endogenous mechanisms over time, taking on secondary maintenance roles. It also explored the evolvability of miRNAs through simulated evolution.

1. MicroRNA (miRNA) is a class of small non-coding RNAs that are
encoded in genomes. The discovery of miRNAs is one of the
biggest breakthrough in molecular biology in the last decade.
MiRNAs have been viewed by many to form an extra regulatory
layer for gene expression. However, the functional role of
miRNAs in the cell and the evolutionary role of miRNAs in
species are still not well understood. In this study, we integrated
the phylogeny, expression and targeting information of miRNAs
to understand their functional and evolutionary roles, with an
emphasis on human miRNAs. The first part of our study is to
classify miRNAs by their conservational levels. We designed a
bootstrap-based analysis pipeline to identify phylogenetically
related miRNAs and classify them into different conservational
categories based on the presence of their homologs in species.
We found that miRNAs of different conservational levels differ
significantly in their expression, targeting and evolution. More
conserved miRNAs are usually expressed more abundantly in
more tissues, target more genes and are less likely to change in
sequence than less conserved miRNAs. In the second part, we
designed the measure of the apparent repression effectiveness
(ARE) of the epistatic effects of miRNAs on the expression of
their target genes. We found that more conserved miRNAs also
have stronger ARE over their target genes. This reflected a trend
in the functional evolution of miRNAs to establish stronger
epistatic control over the expression of their target genes. In the
third part, we studied the contribution of miRNAs to the
evolution of tissue complexity. We proposed two contradictory

2. models differentiating the possible mechanisms of the
involvement of miRNAs in tissue complexity and evaluated the
models with phylogeny, expression and targeting data of human
genes and miRNAs. Our study showed that miRNAs are likely to
have important roles in the initial establishment of new tissue
identities. However, miRNA-mediated regulations may be
replaced gradually by endogenous mechanisms in a process we
defined as "burn-in." As the result, in most current tissues,
miRNAs may just have secondary maintenance roles in gene
regulation. In the fourth part, we studied the evolvability of
miRNAs by simulated evolution. It is much easier to predict
miRNA targets by sequence than to predict protein-protein
interaction by sequence. In this regard, miRNAs can be used as
model systems for hard evolution problems. We explored the
fitness space of miRNA sequences defined by the expression
profiles of potential target genes. Our results suggested that the
balance of a "selfish" force driving miRNAs to target more genes
and a selective force against targeting highly expressed or vital
genes may result in the exhaustion of evolvability of miRNAs in
evolution