Faculty Bibliography

Vertebrate genomes each encode hundreds of micro-RNAs (miRNAs), yet for few of these miRNAs is there empirical evidence as to which mRNA(s) they regulate. Here we report the identification of human lin-28 mRNA as a regulatory target of human miR-125b and its homolog miR-125a. Studies of miR-125b function in mouse P19 embryonal carcinoma cells induced to develop into neurons suggest a role for this regulatory miRNA in mammalian neuronal differentiation, since its increased concentration in these cells contributes to lin-28 downregulation. Within the lin-28 3' untranslated region (UTR) are two conserved miRNA responsive elements (miREs) that mediate repression by miR-125b and miR-125a. Simultaneous deletion of both miREs renders the lin-28 3' UTR almost completely insensitive to these miRNAs, indicating that these two miREs are the principal elements in the lin-28 3' UTR that respond to miR-125. At the 3' end of each element is an adenosine residue that makes a significant contribution to function irrespective of its complementarity to the 5'-terminal nucleotide of miR-125. By contrast to most earlier reports of gene repression by other miRNAs that are imperfectly complementary to their targets, lin-28 downregulation by miR-125 involves reductions in both translational efficiency and mRNA abundance. The decrease in the mRNA concentration is achieved by a posttranscriptional mechanism that is independent of the inhibitory effect on translation

RNase E is an endonuclease that plays a central role in RNA processing and degradation in Escherichia coli. Like its E. coli homolog RNase G, RNase E shows a marked preference for cleaving RNAs that bear a monophosphate, rather than a triphosphate or hydroxyl, at the 5' end. To investigate the mechanism by which 5'-terminal phosphorylation can influence distant cleavage events, we have developed fluorogenic RNA substrates that allow the activity of RNase E and RNase G to be quantified much more accurately and easily than before. Kinetic analysis of the cleavage of these substrates by RNase E and RNase G has revealed that 5' monophosphorylation accelerates the reaction not by improving substrate binding, but rather by enhancing the catalytic potency of these ribonucleases. Furthermore, the presence of a 5' monophosphate can increase the specificity of cleavage site selection within an RNA. Although monomeric forms of RNase E and RNase G can cut RNA, the ability of these enzymes to discriminate between RNA substrates on the basis of their 5' phosphorylation state requires the formation of protein multimers. Among the molecular mechanisms that could account for these properties are those in which 5'-end binding by one enzyme subunit induces a protein structural change that accelerates RNA cleavage by another subunit

RNase G is a homologue of the essential Escherichia coli ribonuclease RNase E. Whereas RNase E plays a key role in the degradation of mRNA and the processing of tRNA and rRNA in E. coli, the biological functions of RNase G appear more limited. We report here that this difference in function is not merely a consequence of the significantly lower cellular concentration of RNase G, but also reflects differences in the intrinsic properties of these ribonucleases, as overproducing wild-type RNase G at a level up to 20 times the usual cellular concentration of RNase E cannot normally compensate for the absence of RNase E in E. coli. Instead, RNase G can sustain significant growth of RNase E-deficient E. coli cells only when it bears an unnatural extension at its amino terminus (e.g. MRKGINM) or carboxyl terminus (e.g. GHHHHHH). These extensions presumably enable RNase G to cleave critically important cellular RNAs whose efficient processing or degradation ordinarily requires RNase E. That extending the amino terminus of RNase G restores growth to E. coli cells lacking RNase E without detectably improving tRNA processing suggests that RNase E is not essential for tRNA production and is required for cell growth because it plays an indispensable role in the maturation or decay of essential E. coli RNAs other than tRNA

In the summary (of Molecular Microbiology (2000) 46(4), 959-969), Lys-112 was inadvertently referred to as Tyr-112

Despite its importance for RNA processing and degradation in Escherichia coli, little is known about the structure of RNase E or its mechanism of action. We have modelled the three-dimensional structure of an essential amino-terminal domain of RNase E on the basis of its sequence homology to the S1 family of RNA-binding domains. Each of the five surface-exposed aromatic residues and most of the 14 basic residues of this RNase E domain were replaced with alanine to determine their importance for RNase E function. All the surface residues essential for cell growth and feedback regulation of RNase E synthesis mapped to one end of the domain. In vitro assays indicate that these essential residues fall into two functionally distinct groups that form discrete clusters on opposite faces of the S1 domain. One group, comprising Phe-57, Phe-67 and Lys-112 [corrected], is of general importance for the ribonuclease activity of RNase E, whereas the other group, comprising Lys-37 and Tyr-60, is entirely dispensable for catalytic activity in vitro. The side-chains of two residues previously identified as sites of temperature-sensitive mutations lie buried directly beneath the surface region defined by Phe-57, Phe-67 and Lys-112 [corrected], which probably enhances RNase E activity by making a crucial contribution to the binding of substrate RNAs. In contrast to the S1 domain, an arginine-rich RNA-binding domain in the carboxyl half of RNase E appears to have a more peripheral role in RNase E function, as it is not required for feedback regulation, cell growth or ribonuclease activity

RNase E is an important regulatory enzyme that governs the principal pathway for mRNA degradation in Escherichia coli. This endonuclease controls its own synthesis via a feedback mechanism in which the longevity of rne (RNase E) mRNA is modulated by a cis-acting sensory element that responds to changes in cellular RNase E activity. Previous research has shown that this element is an RNA stem-loop (hp2) within the 5'-untranslated region of the rne transcript. Here we report studies involving mutational analysis and phylogenetic comparison that have identified the features of rne hp2 important for its function. These comprise an internal loop flanked on one side by a 2-bp stem and a hairpin loop and on the other side by a longer stem whose sequence is inconsequential. A search of bacterial genome sequences suggests that regulation by an hp2-like element may be a unique evolutionary adaptation of the rne transcript that is not shared by other mRNAs

The labile SAUR transcripts from higher plants contain a conserved DST sequence in their 3'-untranslated regions. Two copies of a DST sequence from soybean are sufficient to destabilize reporter transcripts in cultured tobacco cells whereas variants bearing mutations in the conserved ATAGAT or GTA regions are inactive. To investigate the potential for conserved recognition components in mammalian and plant cells, we examined the function of this instability determinant in mouse NIH3T3 fibroblasts and tobacco BY2 cells. In fibroblasts, a tetrameric DST element from soybean accelerated deadenylation and decay of a reporter transcript. However, a version mutated in the ATAGAT region was equally effective in this regard, and a tetrameric DST element from Arabidopsis was inactive. In contrast, the soybean DST element was more active as an mRNA instability element than the mutant version and the Arabidopsis element, when tested as tetramers in tobacco cells. Hence, the plant DST element is not recognized in animal cells with the same sequence requirements as in plant cells. Therefore, its mode of recognition appears to be plant-specific

The endoribonuclease RNase E plays an important role in RNA processing and degradation in Escherichia coli. The construction of an E. coli strain in which the cellular concentration of RNase E can be precisely controlled has made it possible to examine and quantify the effect of RNase E scarcity on RNA decay, gene regulation and cell growth. These studies show that RNase E participates in a step in the degradation of its RNA substrates that is partially or fully rate-determining. Our data also indicate that E. coli growth requires a cellular RNase E concentration at least 10-20% of normal and that the feedback mechanism that limits overproduction of RNase E is also able to increase its synthesis when its concentration drops below normal. The magnitude of the in-crease in RNA longevity under conditions of RNase E scarcity may be limited by an alternative pathway for RNA degradation. Additional experiments show that RNase E is a stable protein in E. coli. No other E. coli gene product, when either mutated or cloned on a multicopy plasmid, seems to be capable of compensating for an inadequate supply of this essential protein

RNA-binding proteins are central to posttranscriptional gene regulation and play an important role in a number of major human diseases. Cloning such proteins is a crucial but often difficult step in elucidating the biological function of RNA regulatory elements. To make it easier to clone proteins that specifically bind RNA elements of interest, we have developed a rapid and broadly applicable in vitro genetic selection method based on T7 phage display. Using hairpin II of U1 small nuclear RNA (U1hpII) or the 3' stem loop of histone mRNA as bait, we could selectively amplify T7 phage that display either the spliceosomal protein U1A or the histone stem loop-binding protein from a lung cDNA phage library containing more than 10(7) independent clones. The use of U1hpII mutants with various affinities for U1A revealed that this method allows the selection even of proteins that bind their cognate RNA targets with relatively weak affinities (K(d) as high as the micromolar range). Experiments with a mixture of recombinant phage displaying U1A or the closely related protein U2B' demonstrated that addition of a competitor RNA can suppress selection of a protein with a higher affinity for a given RNA target, thereby allowing the preferential amplification of a lower affinity protein. Together, these findings suggest that T7 phage display can be used to rapidly and selectively clone virtually any protein that binds a known RNA regulatory element, including those that bind with low affinity or that must compete for binding with other proteins