Stefan Washietl *, Ivo L. Hofacker *, and Peter F. Stadler *^{, 1, @}

*Department of Theoretical Chemistry and Structural Biology, University of Vienna, Währingerstrasse 17, A-1090 Wien, Austria; and
 ¹ Bioinformatics Group, Department of Computer Science, and Interdisciplinary Center for Bioinformatics, University of Leipzig, Härtelstrasse 16-18, D-04107 Leipzig, Germany

^@ To whom correspondence should be addressed:
Peter F. Stadler, E-mail:    studla@bioinf.uni-leipzig.de
http://www.tbi.univie.ac.at/

We report an efficient method for detecting functional RNAs. The approach, which combines comparative sequence analysis and structure prediction, already has yielded excellent results for a small number of aligned sequences and is suitable for large-scale genomic screens. It consists of two basic components: (i) a measure for RNA secondary structure conservation based on computing a consensus secondary structure, and (ii) a measure for thermodynamic stability, which, in the spirit of a z score, is normalized with respect to both sequence length and base composition but can be calculated without sampling from shuffled sequences. Functional RNA secondary structures can be identified in multiple sequence alignments with high sensitivity and high specificity. We demonstrate that this approach is not only much more accurate than previous methods but also significantly faster. The method is implemented in the program RNAZ, which can be downloaded from http://www.tbi.univie.ac.at/~wash/RNAz. We screened all alignments of length n>50 in the Comparative Regulatory Genomics database, which compiles conserved noncoding elements in upstream regions of orthologous genes from human, mouse, rat, Fugu, and zebrafish. We recovered all of the known noncoding RNAs and cis-acting elements with high significance and found compelling evidence for many other conserved RNA secondary structures not described so far to our knowledge.

http://www.tbi.univie.ac.at/papers/SUPPLEMENTS/RNAz

Supporting Information
http://www.pnas.org/cgi/content/full/0409169102/DC1

Files in this Data Supplement:

Supporting Figure 3:
Separation of native alignments (green) from random controls (red) for various classes of ncRNAs.

Fig. 3. Separation of native alignments (green) from random controls (red) for various classes of ncRNAs. The test sets are the same as used in Table 1 with mean pairwise identities between 60% and 100% and two to four sequences per alignment.

10. Hovsepian JA, and Frenster JH, "Bioassays of Isolated Nuclear RNA Species as Activators of DNA Transcription".

9. Kuwabara T, Hsieh J, Nakashima K, Taira K, and Gage FH, "A Small Modulatory dsRNA Specifies the
Fate of Adult Neural Stem Cells".

8. Frenster JH, and Hovsepian JA, "Activator RNA Exchange during Interphase Chromatin Reprogramming".

7. Gottesfeld JM, and Barbas CF III, "RNA as a Transcriptional Activator".

6. Buskirk AR, Landrigan A, and Liu DR, "Engineering a Ligand-Dependent RNA Transcriptional Activator".

5. Buskirk AR, Kehayova PD, Landrigan A, and Liu DR, "In Vivo Evolution of an RNA-Based Transcriptional Activator".

4. Persengiev SP, Zhu X, and Green MR, "Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs)".

3. Lanz RB, Chua SS, Barron N, Söder BM, DeMayo F, and O'Malley BW, "Steroid Receptor RNA
Activator Stimulates Proliferation as Well as Apoptosis In Vivo".

2. Saha S, Ansari AZ, Jarell KA, and Ptashne M, "RNA Sequences that Work as Transcriptional Activating
Regions".

1. Lanz RB, Razani B, Goldberg AD, and O'Malley BW, "Distinct RNA Motifs are Important for
Coactivation of Steroid Hormone Receptors by Steroid Receptor RNA Activator (SRA)".

1. Frenster JH, Allfrey VG, and Mirsky  AE, "Metabolism and Morphology of Ribonucleoprotein Particles from the Cell Nucleus of Lymphocytes" Proc. Natl. Acad. Sci. USA, vol. 46, pp. 432-444 (April, 1960).

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3. Frenster JH, Allfrey VG, and Mirsky, AE, "Repressed and Active Chromatin Isolated from Interphase Lymphocytes", Proc. Natl. Acad. Sci., USA, vol. 50, no. 6, pp. 1026-1032 (Dec. 1963):

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5. Frenster JH, "Nuclear Polyanions as De-Repressors of Synthesis of Ribonucleic Acid", Nature, vol. 206, no. 4985, pp. 680-683 (May 15, 1965).

6. Frenster JH, "A Model of Specific De-repression within Interphase Chromatin", Nature, vol. 206, no. 4990, pp. 1269-1270 (June 19, 1965 ).

7. Frenster JH, "Analysis of Queueing and Renewal within Human Systems", Nature Vol. 207, No. 5002, pp. 1139-1140 (September 11, 1965).

8. Frenster JH, "Localized Strand Separations within Deoxyribonucleic Acid during Selective Transcription", Nature, vol. 208: no. 5013, pp. 894-896 (November 27, 1965).

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11. Herstein PR, and Frenster JH, "Mated Models of Gene Regulation in Eukaryotes".

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14. Frenster JH, "Single-Cell Analysis of DNase I-Sensitive Sites during Neoplastic and Normal Cell Differentiation within Human Bone Marrow".

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A Brief History of Activator RNA:

Links to Euchromatin Activator RNA Reviews:
Links to Euchromatin Activator RNA Research:
Links to Ultrastructural Probes of DNase I-Sensitive Sites:
Links to RNA as a Therapeutic Agent:
Links to Hodgkin Lymphoma Immuno-Pathology:
Links to Activated T-Lymphocyte Immunotherapy:
Links to Medical Systems Biology:
Links to Selective Gene Transcription:
Links to RNA-Induced Epigenetics:
Links to RNA-Induced Embryogenesis:
Links to RNA and Biological Causality:
Links to Reprogramming and Neoplasia:

A Brief History of Activator RNA:

"Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA". (PowerPoint Presentation).

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