The secondary structure of RNA molecules can be studied using experimental, thermodynamical and comparative methods. Programs that calculate the most thermodynamically favorable structure such as mfold (2) produce connection data: a list of bases and of numbers indicating secondary structure interactions. In DCSE (1) the structural information is incorporated in the alignment by interspersing the sequence with special symbols denoting the start and end of structural features. A special "helix numbering line" contains the names for the helix strands, and indicates which are complementary. Although these forms of structural information are very useful, they cannot be used for publications as they are difficult to evaluate. Since the classical 2D drawing of the secondary structure is easier to grasp and more aesthetically pleasing, it is the preferred visualization for publications.
Although several programs (7-12) exist that produce 2D structure drawings, most are too tightly coupled to an energy minimization prediction program to be of general use. Furthermore, the user cannot easily change the produced layout: Much effort has been put into automatically producing a layout where none of the helices overlap, but this often does not properly emphasize similarities in structure because of insertions or deletions in less conserved areas. Other common problems are limitations to the size of molecule that can be displayed, and the inability to handle complex structural elements such as pseudo-knots.
RnaViz is a program for producing publication-ready secondary structure drawings starting from the connection data in the ct format as produced by mfold (2) or alignments with extra structure information in the DCSE format (1). It does not try to produce non-overlapping drawings, so the first drawing produced for a new molecule might show considerable overlap. However, this structure can be easily arranged interactively according to the user's wishes. As illustrated in Fig. 1 and 2, RnaViz is capable of producing large as wel as complicated structures. The layout or skeleton of a structure can be saved to a file, and used as a template to automatically arrange similar structures in the same layout. Skeletons for several molecules are included in the package. The program also incorporates many options for labeling the structure or emphasizing special features in it.
RnaViz needs a modified version of Tcl which has the dash patch (Jan Nijtmans) and some other patches applied. It also makes use of the Extral, Peos and Visexport extensions (Peter De Rijk, unpublished). RnaViz has already been ported to several operating systems; binary distributions of the modified Tcl and the RnaViz package are available for Linux and MS Windows 95. The sources are available for people who want to port the code to other systems.
Files can be selected using a file selection box. Since RnaViz can contain several structures on one page, structures already on the page are not automatically deleted when a new file is opened. However, the page can be cleared before a new structure is loaded. Individual structures on the page can also be deleted.
The type of supported file formats is automatically detected. Opening a file in the RnaViz structure format will cause the structures in the file to be loaded directly onto the current page. When a DCSE alignment file or an mfold ct file is opened, the program will prompt for a skeleton file. If one is given, the program will produce drawings of the structure(s) in the file with the layout given in the skeleton file. If no skeleton is given, the structure drawings produced will probably contain overlapping areas. However, this can be easily fixed interactively. The program distribution contains examples and skeletons of several types of RNA molecules, viz. tRNA, 5S, SSU and LSU rRNA and group I introns. When a DCSE or ct file contains more than one structure, one or more of these can be selected. The user can choose to either draw all selected structures on the current page, or to create several structure files, each containing a drawing of one of the selected structures.
Several user-definable parameters control how a newly created structure will be drawn. Among others, the general distance between bases in single stranded areas, the distance between bases in a helix and between the bases in a base pair can be set. By default, bases in a base pair are connected by a dot according to the IUPAC convention (15), but both width and length of the connections between the bases of standard and non-standard base pairs can be changed independently. The bases of non-standard base pairs can also be made to bulge slightly out of the helix. These settings can be changed for a drawn structure, but will only have effect when the structure or parts of it are redrawn. It is also possible to scale a structure.
Under MS Windows, the drawings can be printed directly using the standard Windows printer drivers, or exported to the clipboard for further processing in other packages. On Unix systems postscript files are produced that can either be printed directly to a postscript printer, or to other printers using ghostscript.
Usually RnaViz is used in the "element selection" mode. In this mode, clicking on a base that belongs to a helix will select the entire helix. A structure on the page can be rearranged quickly by clicking on a base or on the selection and dragging it to a different position. When the selection is released, the bases connecting the selection to the rest of the structure will be rearranged so as to maintain a correct structure drawing. If the distance between the bases reaches a given threshold, they will be automatically connected by a line. The threshold and the width of the line can be chosen by the user. The alternate mouse button can be used to select the apical portion of a helix, starting from the segment clicked on, rather than a complete helix. In DCSE files helix segments are defined as the parts of a helix separated by internal or bulge loops. Using the "Select tree" entry in the "Edit" menu or its key shortcut the entire area enclosed by the two strands of the selected helix can be added to the selection, e.g. the tree starting from helix D1 in Fig. 1 contains all helices from D2 to D22. The selected part of a structure can be rotated by dragging with the "Shift" key pressed. The center of rotation is indicated by a gray circle, and can be repositioned by clicking with the alternate mouse button while holding the Shift key. The selection can also be oriented into a specific direction using the "Orient helix" or "Orient" options in the "Geometry" menu. When more than one helix is selected, the last helix added will determine the orientation. The "Geometry" menu also offers options to easily straighten or bend the single stranded areas, or to flip a helix. The latter is often necessary to create a clear drawing of pseudoknots as shown in Fig. 2. As illustrated in Fig.1, areas with unknown structure can be drawn unformatted.
In contrast to the previous mode, the "single select" mode allows individual selection and positioning of objects. Only the selected objects will be moved, without redrawing the objects connecting them to the rest of the structure. This makes any special arrangement of objects possible. Other selection modes are the "select tree" and "select sub-element", which automatically select a tree or a set of segments of a helix.
RnaViz also contains a limited drawing component.
Several types of objects such as texts, rectangles, ovals, lines
and polygons can be created and edited. Any of these objects can
be used as a label by linking them to a certain base. An example
of several types of labels is shown in Fig. 3 of a tRNA.
A special type of property are tags. A tag is a short
text that is attached to an object. Every object can have several
tags. The tags attached to an object can be changed individually
by selecting the object and invoking the "Edit tags"
dialog. Tags can be added to or removed from groups of objects
using the object configuration dialog. If the structure was created
starting from a DCSE alignment that contained a line named "mask"
each base is tagged with the character on the corresponding position
in the "mask" line. It is also possible to add a list
of tags to the sequence. Tags are a very powerful feature: they
can be used to indicate any special feature of specific bases,
such as the variability of their position (16) or their use in
a certain analysis. Since object configuration can be limited
to objects with certain tags, it is easy to display such bases
using different colors or fonts as demonstrated in Fig. 4.
RnaViz solves these problems. Secondary structure
drawings can be produced from data produced by other programs,
without the need to enter the sequences from the keyboard. Rearrangement
of the structure is straightforward, and several methods for annotating
or labeling structures are present. The use of Tcl/Tk for the
interface makes the program highly portable and extensible, thus
more generally useful. In the future the algorithm for creating
the initial layout could be improved, as drawings created without
skeleton usually contain overlapping areas. However, the easy
way structures can be rearranged and the use of skeleton files
make this a minor issue.
Figure 1. Secondary structure model of the large subunit ribosomal RNA of Xenopus leavis. The areas enclosed by helices C1 and E20 have been drawn unstructured.
Figure 2. Drawing of the group I intron in the large subunit rRNA gene of Tetrahymena termophila (3). Helices P3 and P7 form a pseudoknot structure. Helices P7, P8, P9 P9_1 and P9_2 have been flipped in order to draw this structure properly. The bases drawn in italics are part of the exons bordering the intron.
Figure 3. The interface of RnaViz running on MS Windows 95. All RnaViz functions can be invoked from the menubar at the top of the Window. The window shows the structure of yeast phenylalanine tRNA. Several labels indicate base numbers and special areas in this molecule. The blue o's at the termini are used for the orientation of the terminal single strands, but do not appear in print. Contrary to the structures in Fig. 1 and 4, the sequence is drawn anti clockwise, as is customary for tRNAs.
Figure 4. Secondary structure model of E.coli SSU rRNA, where the variablity of each position (16) is indicated by the color of the base according to the scale at the bottom of the page. The bases at the most variable positions are colored red, while those at the the most conserved positions are indicated in black. Gray is used positions where variability could not be measured.