Functional RNAs

Most eukaryotic RNAs are synthesized as precursors that contain excess sequences which are removed prior to the generation of mature, functional RNA. 

Transcription is catalyzed by DNA-dependent RNA polymerases. These act in a similar way to DNA polymerases (see p. 240), except that they incorporate ribonucleotides instead of deoxyribonucleotides into the newly synthesized strand also, they do not require a primer. Eukaryotic cells contain at least three different types of RNA polymerase. RNA polymerase I synthesizes an RNA with a sedimentation coef cient (see p. 200) of 45 S, which serves as precursor for three ribosomal RNAs. The products of RNA polymerase II are hnRNAs, from which mRNAs later develop, as well as precursors for snRNAs. Finally, RNA polymerase III transcribes genes that code for tRNAs, 5S rRNA, and certain snRNAs. These precursors give rise to functional RNA molecules by a process called RNA maturation (see p. 246). Polymerases II and III are inhibited by a-amanitin, a toxin in the Amanita phalloides mushroom. 

The large subimit of RNA polymerase II plays an important role at the beginning of the transcription process. The large subimit of the mammalian enzyme contains 52 copies of the heptamer sequence YSPTSPS in the C-terminal domain (CTD) at which phosphorylation occurs. Phosphorylation occurs extensively on the Ser-residues of the CTD, to a lesser degree at the Thr-residues, and, very rarely, at the Tyr-residues. Two forms of RNA polymerase II can be isolated from cellular extracts a underphosphory-lated form and a hyper-phosphorylated form. The isoforms fulfill different functions RNA polymerase found in the initiation complex tends to display little or no phosphorylation at the C-terminus of the large subunit, while RNA polymerase II active in elongation is hyperphosphorylated in this region of the protein. 

Messenger RNA is only one of several classes of cellular RNA. Transfer RNAs serve as adapter molecules in protein synthesis covalently linked to an amino acid at one end, they pair with the mRNA in such a way that amino acids are joined to a growing polypeptide in the correct sequence. Ribosomal RNAs are components of ribosomes. There is also a wide variety of special-function RNAs, including some (called ribozymes) that have enzymatic activity. All the RNAs are considered in detail in Chapter 26. The diverse and often complex functions of these RNAs reflect a diversity of structure much richer than that observed in DNA molecules. 

One definition is the DNA (or RNA) sequences necessary to produce a peptide (or RNA). Some viruses have RNA as their genetic material and some genes do not code for a protein but make a functional RNA such as tRNA or rRNA. 

Functional RNA molecules, whether natural or produced in the lab through directed evolution, typically require distinctive secondary structures to fulfill their function for a nice example we refer to Schwienhorst [8]. These structures serve as a scaffold that allows the formation of, e. g., a catalytic site. Thus, sequence constraints observed in RNA molecules selected for a particular function, such as binding or catalysis, may be due to direct involvement in that function or due to stabilization of the structure. Predicted RNA secondary structures can be most helpful to identify such structural constraints and to interpret the results of a directed evolution experiment in terms of structure-function relationships. 

In nature, to preserve function a strong selective pressure may act on the secondary structure of functional RNA molecules, while the sequences diverge. This effect... 

SAXS and Conformational Changes in Small Functional RNAs 238... 

This kind of observation has led to the idea that functional RNA molecules tend to fold an order of magnitude more slowly than proteins, as kinetic traps may form on the folding pathway which then take time to unfold again so that the molecule can finally reach its functional folded state. However, the lack of time-resolved RNA folding measurements to date on a range of different functional RNAs means that this idea has not yet been substantiated in depth. 

Ngo, V.N. et al. 2006. A loss-of-function RNA interference screen for molecular targets in cancer. Nature 441, 106-110. 

Modified nucleosides incorporated into small RNA model systems can also be used to investigate the global versus individual effects of modified nucleotides on natural RNAs, such as rRNA or tRNA. For example, in some early studies, Yarian et al. (44) demonstrated that pseudouridine (Table 1) leads to increased thermal stability of the tRNA anticodon stem-loop region. Later, Meroueh et al. (45) demonstrated that pseudouridines have opposing effects on rRNA helix 69 stability, which depends on their specific locations and sequence contexts. These effects on stability may be important for conformational switching mechanisms in functional RNAs (46, 47). 

N.A. Woychik. 1998. Fractions to functions RNA polymerase II thirty years later Cold Spring Harbor Symp. Quant. 

RNA molecule that are necessary to produce a fully functional RNA. 

VVoychik, N. A, 1998. f ractions to functions RNA polymerase JI thirty years later, Coid Spring Har/ior Symp. Quant. Biol. 63 311 317. 

The rationale of this experiment was that active RNA polymerase II in the onecell embryo would complement the inhibited RNA polymerase II present in the transplanted two-cell nucleus, which brings with it all of the other transcription factors required for the expression of the TRC. Results of these experiments indicated that TRC expression was readily observed in G2 of the one-cell embryo. Moreover, the level of expression was 30 to 40% that observed when two-cell embryos were radiolabeled for the same length of time. Thus, the one-cell embryo contains functional RNA polymerase II, that is, it is transcriptionally competent. 

Functional RNA polymerase I and III are also present in the one-cell embyro (Nothias et al., 1996). Injection of a chloramphenical acetyl transferase reporter gene under the control of the RNA polymerase I-dependent ribosomal DNA promoter into the male pronucleus of S phase-arrested, one-cell embryos (the embryos were incubated in the presence of aphidicolin, which inhibits DNA polymerases a and 8) revealed accumulation of the appropriate transcript by G2 of the one-cell embryo. The amount of this transcript was about 20% of that maximally accumulated when the cleavage-arrested embryos were cultured to a time that corresponded chronologically to the two-cell stage and then were analyzed for expression. A similar result was obtained when the S phase-arrested, one-cell embryos were injected with a plasmid bearing the RNA polymerase Ill-dependent adenovirus VAl RNA gene. In this case, the amount of transcript accumulated by G2 of the S phase-arrested, one-cell embryo was around 30% of that maximally accumulated. 

Using RNAz, it is possible to efficiently screen alignments for functional RNA secondary structures. It is important to note that RNAz cannot distinguish functional RNA elements that are part of ncRNAs from elements that are m-regulatory elements of mRNAs. 

Using these tools, the user will find, for example, that in the random control 1.0 % of the input sequences are predicted as RNA on the p > 0.9 level. This is exactly the false-positive rate as expected (Subheading 3.4.3.). The absolute number of false-positives, however, strongly depends on the specific screen. In this example we have 88 hits p > 0.9 without RNA annotation and find that 39 hits should be expected by chance. So we must expect that roughly half of our predictions are false-positives. On the other hand, this implies that the other half of the predicted loci should be real functional RNA structures, either as part of a ncRNA or as regulatory element of a mRNA. However, one always have to bear in mind possible shortcomings of this kind of random control (see Note 6). 

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