database of RNA elements
regulating nuclear RNA export in mammals
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  • Rationale
  • Content description
  • Setting the parameters for the alignment
  • How to interpret the returned alignments
  • How to cite ExportAid
  • RATIONALE
    Regulation of mRNA nuclear export or retention is not yet well understood. In particular, some RNA cis-acting elements promote or prevent the export of transcripts that do not undergo splicing so would not be able to exit the nucleus spontaneously. Some of these elements are adopted also from retrovirus in order to express their unspliced mRNAs in the host cell. To collect of all the RNA sequences involved in nuclear export, and of their trans-acting elements is important for a better understanding of the transcript's nuclear export process and functions. By hand-curated literature screening we collected, in ExportAid database, experimentally assessed data about RNA elements regulating nuclear export or retention of endogenous, heterologous or artificial RNAs in mammalian cells. We anotated also information on functional sequences, their genomic location, experimental data and involved export pathways. This database could help to understand the rules of the RNA export language and to study the possible export efficiency alterations due to mutations or polymorphisms. ExportAid is freely accessible at www.introni.it/ExportAid/ExportAid.html and, up to date, it stores 235 and 96 RNA elements respectively increasing and decreasing export efficiency, and 98 neutral assessed sequences.


    CONTENT DESCRIPTION:

    Export Identifier
    This is an unique identifier assigned to each ExportAid record.

    Species the sequence belongs to
    The sequences can derive from viral or mammalian genomes, or be artificial, such as mutated or antisense constructs.

    RNA functional element
    The functional elements lying in assessed sequences are specified here. Some of them do not contain any known element and can be neutral for export or unexpectedly functional.

    Sequence structure investigated
    This field describes, in detail, the structure of constructs. They can derive from a genome or be produced by joining sequences from different parts of a genome or from different genomes. Some constructs put together different functional elements in order to evaluate their interaction. Moreover, here it is shown if the sequences belong to genes.

    Construct name
    As usual, constructs are cited in papers by the name, in order to make their recognition easier, we collected their name. When the reference paper did not indicate this name we annotated at least the name of the used vector.

    Cell line used
    We reported the cell lines used to assess the functional effect of a sequence so the user can observe its spectrum of validity.

    Score
    We assigned a score ranging from -10 to +10 in order to describe the effect of an RNA element in terms of tendency to increase or decrease the nuclear export efficiency. Export efficiency of a transcript is usually defined as the ratio between the amount of RNA found in cytoplasm and the total RNA. In particular, we have indicated "(0) neutral effect" when a reference paper showed that the insertion of a functional element in a construct did not alter the export efficiency of that construct. We assigned a positive or a negative score to the functional elements that respectively increased or decreased the RNA export efficiency. The user has to consider that a score of -10 does not mean that the sequence was completely retained in the nucleus. Instead it means that the sequence concerned had the maximum effect, among/over the sequences assessed in the reference paper, in terms of decrease of the export efficiency. The same goes for the score +10, it does not mean that the RNA sequence is completely exported into cytoplasm but only that it had the maximum effect in increasing the export. The user has to take into account that the score allows to compare the strength of functional elements only of the same reference paper. Moreover it should be noted that some elements have an enhancer or inhibitory effect depending on the presence or not of their trans-acting element. For example, the RRE element of HIV prevents or enhances nuclear export respectively in absence or in presence of its Rev protein. We annotated also this kind of context-dependent functionality.

    Coordinates
    This field shows the sequence coordinates according to a particular GenBank accession number. Since we realized that the same viral sequence can be associated to different genomic assembly, in some ExportAid records we have reported genomic coordinates according to different GenBank accession numbers. For example, the HIV genome was mainly referred to by K03455 and AF033819 NCBI IDs but their coordinates are slid/displaced/shifted of 455 nt so we have reported both notations. Users can observe that also in the field “Sequence structure investigated” there are some coordinates and sometimes they differ from the genomic coordinates reported in the “Note” field. This is because in the former field we have reported the numeration used in the reference paper which, for convenience, could be referred to the start of a construct or to the coding sequence or of the mRNA.

    Note
    It specifies if the experiment was performed in presence of a RNA trans-acting element or other experimental details.

    References
    Here we report the PubMed Identifier of the reference paper the sequence was derived from.

    RNA binding protein
    The "RNA binding protein" field shows which RNA export factor is known to bind the cis-acting element. We indicated "None" when the sequence did not contain any known functional element and was neutral (sequence which insertion or deletion in a construct did not alter the export efficiency) for export according to the literature. In this case, we assumed that no RNA binding protein involved in export could bind this sequence. Conversely, we indicated "Unknown" when the sequence was assessed as functional therefore it should bind some not yet identified RNA binding proteins involved in export.

    Export pathway
    The "Export pathway" field shows the pathway involved in nuclear export of that sequence. When this information did not appear in the reference paper in which the element functionality was assessed, we attempted to derive it from well-established knowledge. We indicated "None" when the sequence did not contain any known functional element and was neutral (sequence which insertion or deletion in a construct did not alter the export efficiency) for export according to the literature. In this case, we assumed that no RNA binding protein involved in export could bind this sequence. Conversely, we indicated "Unknown" when the sequence was assessed as functional but the involved export pathway was not yet identified.

    Sequence
    It shows the exact sequence assessed in the reference paper and it should be taken into account that often not the whole sequence is functional but it harbours one or more shorter functional elements. To help the user to identify the functional elements inside a sequence we equipped each record with an image that we made and which shows the structure and the genomic context each sequence derives from. The colour of the boxes representing functional elements reflects their main activity and it does not take into account that an element can exert opposite function depending on the presence or absence of its trans-acting element. For example, we coloured the INS element of HIV in red because usually it prevents export and the RRE element of HIV in green because generally it promotes export.

    RNA folding predictions
    It contains links to submit ExportAid sequence to some RNA folding prediction tools.


    SETTING THE PARAMETERS FOR THE ALIGNMENT:

    E value
    This setting specifies the statistical significance threshold for reporting matches against database sequences. The default value (10) means that 10 such matches are expected to be found merely by chance, according to the stochastic model of Karlin and Altschul (1990). If the statistical significance ascribed to a match is greater than the EXPECT threshold, the match will not be reported. Lower EXPECT thresholds are more stringent, leading to fewer chance matches being reported.

    Word size
    BLAST is a heuristic that works by finding word-matches between the query and database sequences. One may think of this process as finding "hot-spots" that BLAST can then use to initiate extensions that might eventually lead to full-blown alignments. For nucleotide-nucleotide searches (i.e., "blastn") an exact match of the entire word is required before an extension is initiated, so that one normally regulates the sensitivity and speed of the search by increasing or decreasing the word-size. For other BLAST searches non-exact word matches are taken into account based upon the similarity between words. The amount of similarity can be varied so one normally uses just the word-sizes 2 and 3 for these searches.

    Low complexity filter
    This function mask off segments of the query sequence that have low compositional complexity, as determined by the SEG program of Wootton and Federhen (Computers and Chemistry, 1993) or, for BLASTN, by the DUST program of Tatusov and Lipman. Filtering can eliminate statistically significant but biologically uninteresting reports from the blast output (e.g., hits against common acidic-, basic- or proline-rich regions), leaving the more biologically interesting regions of the query sequence available for specific matching against database sequences.
    We recommend to set this value to "No" because ExportAid database collects some sequences made of tandem copies of a motif.

    Gap open penalty
    Cost to open a gap. Increasing the gap openning penalty will decrease the number of gaps in the final alignment.

    Gap extend penalty
    Cost to extend a gap. Increasing the gap extension penalty favors short gaps in the final alignment, conversly decreasing the gap extension penalty favors long gaps in the final alignment.


    HOW TO INTERPRET THE RETURNED ALIGNMENTS:
    We have installed local NCBI BLAST to allow the user, via the Blast page, to search his sequence versus the entire database. In this case, the returned alignments have to be correctly interpreted because not all alignments mean the presence of an RNA functional element in the user sequence. As previously mentioned, ExportAid collects the exact sequences assessed in the reference papers so some of them consist of hundreds or thousands of nucleotides but only a shorter part is thought to be functional. So the user should verify if the returned alignment overlaps to a known functional element by using the images enclosed with each record. In particular, the entire record and the image of sequence element can be visualized by the two buttons beside each returned alignment. Analogously, if an alignment does not extend to the entire length of the functional element it does not guarantee the functionality of the user submitted sequence, since the binding site for its trans-acting element could be lacking or its binding could be prevented. In fact, also RNA structure are important for the function and a shorter sequence would not fold correctly.


    HOW TO CITE EXPORTAID:
    ExportAid: database of RNA elements regulating nuclear RNA export in mammals.
    Giulietti M, Milantoni SA, Armeni T, Principato G, Piva F.
    Bioinformatics. 2015; 31(2):246-251.
    PMID: 25273107











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    Computational Biology Group
    Polytechnic University of Marche, Italy | 2014