Increased efficiency of evolved group I intron spliceozymes by decreased side product formation.

The group I intron ribozyme from Tetrahymena was recently reengineered into a trans-splicing variant that is able to remove 100-nt introns from pre-mRNA, analogous to the spliceosome. These spliceozymes were improved in this study by 10 rounds of evolution in Escherichia coli cells. One clone with increased activity in E. coli cells was analyzed in detail. Three of its 10 necessary mutations extended the substrate binding duplexes, which led to increased product formation and reduced cleavage at the 5'-splice site. One mutation in the conserved core of the spliceozyme led to a further reduction of cleavage at the 5'-splice site but an increase in cleavage side products at the 3'-splice site. The latter was partially reduced by six additional mutations. Together, the mutations increased product formation while reducing activity at the 5'-splice site and increasing activity at the 3'-splice site. These results show the adaptation of a ribozyme that evolved in nature for cis-splicing to trans-splicing, and they highlight the interdependent function of nucleotides within group I intron ribozymes. Implications for the possible use of spliceozymes as tools in research and therapy, and as a model for the evolution of the spliceosome, are discussed.

Spliceozymes: ribozymes that remove introns from pre-mRNAs in trans.

Group I introns are pre-mRNA introns that do not require the spliceosome for their removal. Instead, they fold into complex three-dimensional structures and catalyze two transesterification reactions, thereby excising themselves and joining the flanking exons. These catalytic RNAs (ribozymes) have been modified previously to work in trans, whereby the ribozymes can recognize a splice site on a substrate RNA and replace the 5'- or 3'-portion of the substrate. Here we describe a new variant of the group I intron ribozyme from Tetrahymena that recognizes two splice sites on a substrate RNA, removes the intron sequences between the splice sites, and joins the flanking exons, analogous to the action of the spliceosome. This 'group I spliceozyme' functions in vitro and in vivo, and it is able to mediate a growth phenotype in E. coli cells. The intron sequences of the target pre-mRNAs are constrained near the splice sites but can carry a wide range of sequences in their interior. Because the splice site recognition sequences can be adjusted to different splice sites, the spliceozyme may have the potential for wide applications as tool in research and therapy.

Low selection pressure aids the evolution of cooperative ribozyme mutations in cells.

Understanding the evolution of functional RNA molecules is important for our molecular understanding of biology. Here we tested experimentally how two evolutionary parameters, selection pressure and recombination, influenced the evolution of an evolving RNA population. This was done using four parallel evolution experiments that employed low or gradually increasing selection pressure, and recombination events either at the end or dispersed throughout the evolution. As model system, a trans-splicing group I intron ribozyme was evolved in Escherichia coli cells over 12 rounds of selection and amplification, including mutagenesis and recombination. The low selection pressure resulted in higher efficiency of the evolved ribozyme populations, whereas differences in recombination did not have a strong effect. Five mutations were responsible for the highest efficiency. The first mutation swept quickly through all four evolving populations, whereas the remaining four mutations accumulated later and more efficiently under low selection pressure. To determine why low selection pressure aided this evolution, all evolutionary intermediates between the wild type and the 5-mutation variant were constructed, and their activities at three different selection pressures were determined. The resulting fitness profiles showed a high cooperativity among the four late mutations, which can explain why high selection pressure led to inefficient evolution. These results show experimentally how low selection pressure can benefit the evolution of cooperative mutations in functional RNAs.

St. John's Wort inhibits insulin signaling in murine and human adipocytes.

Adipocytes are insulin-sensitive cells that play a major role in energy homeostasis. Obesity is the primary disease of fat cells and a major risk factor for the development of Type 2 diabetes, cardiovascular disease, and metabolic syndrome. The use of botanicals in the treatment of metabolic diseases is an emerging area of research. In previous studies, we screened over 425 botanical extracts for their ability to modulate adipogenesis and insulin sensitivity. We identified St. John's Wort (SJW) extracts as inhibitors of adipogenesis of 3T3-L1 cells and demonstrated that these extracts also inhibited insulin-sensitive glucose uptake in mature fat cells. In these follow-up studies we have further characterized the effects of SJW on insulin action in both murine and human fat cells. We have shown that SJW also attenuates insulin-sensitive glucose uptake in human adipocytes. Moreover, SJW inhibits IRS-1 tyrosine phosphorylation in both murine and human fat cells. Botanical extracts are complex mixtures. Many bioactive compounds have been identified in SJW, including hypericin (HI) and hyperforin (HF). We have examined the ability of HI and HF, purified from SJW, to modulate adipocyte development and insulin action in mature adipocytes. Our novel studies indicate that the profound effects of SJW on adipogenesis, IRS-1 activation, and insulin-stimulated glucose uptake are not mediated by HI and/or HF. Nonetheless, we propose that extracts of SJW may contribute to adipocyte related diseases by limiting differentiation of preadipocytes and significantly inducing insulin resistance in mature fat cells.

St. John's Wort inhibits adipocyte differentiation and induces insulin resistance in adipocytes.

Adipocytes are insulin sensitive cells that play a major role in energy homeostasis. Obesity is the primary disease of fat cells and a major risk factor for the development of Type II diabetes, cardiovascular disease, and metabolic syndrome. Obesity and its related disorders result in dysregulation of the mechanisms that control adipocyte gene expression and function. To identify potential novel therapeutic modulators of adipocytes, we screened 425 botanical extracts for their ability to modulate adipogenesis and insulin sensitivity. We observed that less than 2% of the extracts had substantial effects on adipocyte differentiation of 3T3-L1 cells. Two of the botanical extracts that inhibited adipogenesis were extracts from St. John's Wort (SJW). Our studies revealed that leaf and flower, but not root, extracts isolated from SJW inhibited adipogenesis as judged by examining PPARgamma and adiponectin levels. We also examined the effects of these SJW extracts on insulin sensitivity in mature 3T3-L1 adipocytes. Both leaf and flower extracts isolated from SJW substantially inhibited insulin sensitive glucose uptake. The specificity of the observed effects was demonstrated by showing that treatment with SJW flower extract resulted in a time and dose dependent inhibition of insulin stimulated glucose uptake. SJW is commonly used in the treatment of depression. However, our studies have revealed that SJW may have a negative impact on adipocyte related diseases by limiting differentiation of preadipocytes and significantly inducing insulin resistance in mature fat cells.