UTRdb and UTRsite: specialized databases of sequences and functional elements of 5′ and 3′ untranslated regions of eukaryotic mRNAs. Update 2002

Abstract

The 5′- and 3′-untranslated regions (5′- and 3′-UTRs) of eukaryotic mRNAs are known to play a crucial role in post-transcriptional regulation of gene expression modulating nucleo-cytoplasmic mRNA transport, translation efficiency, subcellular localization and stability. UTRdb is a specialized database of 5′ and 3′ untranslated sequences of eukaryotic mRNAs cleaned from redundancy. UTRdb entries are enriched with specialized information not present in the primary databases including the presence of nucleotide sequence patterns already demonstrated by experimental analysis to have some functional role. All these patterns have been collected in the UTRsite database so that it is possible to search any input sequence for the presence of annotated functional motifs. Furthermore, UTRdb entries have been annotated for the presence of repetitive elements. All Internet resources we implemented for retrieval and functional analysis of 5′- and 3′-UTRs of eukaryotic mRNAs are accessible at http://bighost.area.ba.cnr.it/BIG/UTRHome/.

INTRODUCTION

The completion of the sequencing of human and of other organism genomes has opened new avenues for understanding the basic mechanisms of cell function. These processes mostly rely on a spatial–temporal coordinated expression of genes mediated by regulatory elements embedded in the non-coding part of the genomes. Among non-coding regions, the 5′- and 3′-untranslated regions (5′- and 3′-UTRs) of eukaryotic mRNAs have often been experimentally demonstrated to contain sequence elements crucial for many aspects of gene regulation and expression (1–7).

The main functional roles so far demonstrated for 5′- and 3′-UTR sequences are: (i) control of mRNA cellular and subcellular localization (4,7–9); (ii) control of mRNA stability (1,10,11); (iii) control of mRNA translation efficiency (12–14).

Several regulatory signals have already been identified in 5′- or 3′-UTR sequences, usually corresponding to short oligonucleotide tracts, also able to fold in specific secondary structures, which are protein binding sites for various regulatory proteins.

The analysis of large collections of functionally equivalent sequences (15,16), such as 5′- and 3′-UTR sequences, could indeed be very useful for defining their structural and compositional features as well as for searching the alleged function-associated sequence patterns (17–19). For this reason we constructed UTRdb, a specialized sequence collection, deprived from redundancy, of 5′- and 3′-UTR sequences from eukaryotic mRNAs.

UTRdb entries have been enriched with specialized information, not present in the primary databases, including the presence of sequence patterns demonstrated by experimental evidence to play some functional role. Additionally, because ∼10% of mammalian mRNAs contain repetitive elements in their UTRs (20) but they are usually not annotated in the original records, we decided to add this information into our database as well.

We also created UTRsite, a collection of functional sequence patterns located in the 5′- or 3′-UTR sequences which could prove very useful for automatic annotation of anonymous sequences generated by sequencing projects as well as for finding previously undetected signals in known gene sequences.

UTRdb GENERATION

The specialized database of UTR sequences was generated by UTRdb_gen, a computer program we devised for this task. Eight sequence collections were generated for both 5′- and 3′-UTR sequences, one for each of the eukaryotic division of the EMBL/GenBank nucleotide database, namely: (i) human, (ii) rodent, (iii) other mammal, (iv) other vertebrate, (v) invertebrate, (vi) plant, (vii) fungi and (viii) virus.

UTRdb_gen, performing an accurate parsing of the Feature Table of the relevant EMBL entries is able to automatically generate the various UTRdb collections. Although the feature keys ‘5′UTR’ and ‘3′UTR’ is a valid feature for the EMBL/GenBank entries, only a small percentage of the entries are adequately annotated. Indeed, of the about 250 000 primary entries where UTRdb_gen was able to extract 5′- or 3′-UTR sequences, only 12% contained the 5′UTR or 3′UTR feature key in the corresponding EMBL entry. UTRdb_gen is able to define UTRs, even when these keys are not reported in the primary entry by using a predefinite syntactic parsing of other relevant feature keys, such as mRNA, CDS, exon, intron, etc.

UTRdb_gen automatically annotates generated UTR entries by adding some specialized information such as completeness or incompleteness of the UTR, number of spanned exons and cross-referencing to the primary database entry. A cross reference between 5′- and 3′-UTR sequences from the same mRNA has also been established.

A further interface between the UTRdb_gen and the BLAST engine (parameters: expect < 10^–5, minimum length = 50 nt, percentage identity > 95%) adds information about the position and the identity of any vector that may contaminate UTR entries.

The generation of UTR entries cleaned from redundancy has been obtained by using CLEANUP program (21) which is able to generate automatically, and very quickly, cleaned collections by removing entries that have a similarity and overlapping degree with longer entries present in the database above a user-fixed threshold. In this case, the cut-off parameters we used for the CLEANUP application were 95% for similarity and 90% for overlapping.

The specialized information included in UTR entries is generated by using two programs: (i) UTRnote including information about the location of experimentally defined patterns collected in UTRsite and (ii) UTRrepeat (which uses RepeatMasker) including repetitive elements present in the Repbase database (19). The UTRsite entries describe the various regulatory elements present in UTRs whose functional role has been established on an experimental basis. Each UTRsite entry is constructed on the basis of information reported in the literature and revised by distinguished scientists experimentally working on the functional characterization of the relevant UTR regulatory element.

CONTENT OF UTRdb

Table 1 reports a summary description of UTRdb (release 15.0) which in total contains 247 548 entries and 64 060 991 nt. On average >35% of entries resulted to be redundant and were then removed from the database. Vector contamination was found in 188 and 196 entries of 5′- and 3′-UTRs, respectively.

Table 1. Number of entries (N) and nucleotide length (L) of UTRdb collections (release 15.0) after redundancy cleaning.

Collection
			Redundancy
	N	L	%N	%L
5′-UTR
Fungi	2223	275 886	27.76	16.74
Human	30 922	4 515 966	40.06	22.33
Invertebrate	19 947	2 987 661	28.06	18.60
Other_mammal	5751	852 910	35.51	14.36
Other_vertebrate	7327	792 573	8.61	15.00
Plant	17 819	1 490 067	25.64	13.51
Rodent	19 759	2 518 594	36.76	20.22
Virus	14 663	3 402 809	81.71	73.82
Total	118 411	16 836 466	–	–
3′-UTR
Fungi	2304	465 396	14.84	11.30
Human	36 015	18 906 357	41.72	29.83
Invertebrate	18 230	5 151 363	36.74	20.92
Other_mammal	6927	2 548 434	29.78	17.55
Other_vertebrate	8528	3 351 751	21.22	13.37
Plant	21 526	4 328 226	16.93	13.28
Rodent	21 489	9 113 464	37.32	23.30
Patent	14 118	3 359 534	77.40	69.42
Total	129 137	47 224 525	–	–

UTRdb 15.0 was generated from EMBL release 67. Relevant redundancy percentages calculated with respect to the number of entries (%N) and to the nucleotide length (%L) are also indicated.

5′-UTR sequences were defined as the mRNA region spanning from the cap site to the starting codon (excluded), whereas 3′-UTR sequences were defined as the mRNA region spanning from the stop codon (excluded) to the poly(A) starting site.

A sample entry of UTRdb is shown in Figure 1. The UTRdb entries have been formatted according to the EMBL database format.

Figure 1.

Sample entry of UTRdb. Specialized information not present in the primary EMBL/GenBank database is shown in bold with active crosslinks with other databases underlined. The ‘UT’ line reports information regarding the relevant UTR entry (e.g. complete or partial) as well as the number of spanned exons in the case of genomic DNA sequences. The presence in this sequence entry of a ‘5′ ribosomal mRNA TOP’ (33–35) (UTRsite entry: U0010) and of a microsatellite element has been also annotated.

Table 2 reports functional patterns and repetitive elements included in UTRsite. More entries will be included in further releases. A sample UTRsite entry is reported in Figure 2. Functional patterns, defined on the basis of the information reported in the literature and/or advice by the scientists expert in the field, were described by using the pattern description syntax used in PatSearch program (22).

Table 2. Functional patterns included so far in UTRsite. For each pattern the number of hits with non-redundant UTRdb entries is also reported.

Functional patterns	Reference	Hits found in UTRdb 15.0
Iron-responsive element (IRE)	(24)	110
Histone 3′-UTR stem–loop structure	(25)	38
AU-rich class II destabilising element	(26)	66
Tra-2 and GLI translational regulation element (TGE)	(27)	81
Selenocysteine insertion sequence (SECIS)	(28–30)	2002
Amyloid precursor protein 3′-UTR stability control element	(31)	15
Cytoplasmatic polyadenylation element (CPE)	(32)	5184
Nanos translation control element		1
Ribosomal protein mRNA 5′ TOP	(33–35)	269
TNF mRNA translation repression element	(36)	8
Vimentin 3′-UTR mRNA element	(37)	6
GLUT1 mRNA stabilising element	(38)	66
Internal ribosome entry site (IRES)	(39)	7353
5′-UTR Msl-2	(40)	5
3′-UTR Msl-2	(40)	18
RpmS12 translational control element	(41)	2
Bruno responsive element (BRE)	(42,43)	196
Barley yellow dwarf virus (BYDV) element	(44)	6
ADH 3′-UTR down-regulation control element	(45)	61
15-LOX-DICE	(46)	90
Upstream ORFs	(47)	27 897
Repetitive elements		38 823

Figure 2.

Sample entry of UTRsite describing the ‘Iron responsive element (IRE)’ (24). The IRE functional pattern, which consists of both primary and secondary structure information, is described in the ‘Pattern’ section according to the format adopted by PatSearch program (22).

AVAILABILITY OF UTRdb

UTRdb and UTRsite are publicly available by anonymous FTP (ftp://area.ba.cnr.it/pub/embnet/database/utr/). All internet resources we implemented for retrieval and functional analysis of 5′- and 3′-UTR sequences are accessible at http://bighost.area.ba.cnr.it/BIG/UTRHome/. These include SRS retrieval (23) of UTRdb and UTRsite, also available at the EBI World Wide Web server (http://srs.ebi.ac.uk:80/), UTRscan and UTRblast. The UTRscan utility allows the enquirer to search user submitted sequences for any of the patterns collected in UTRsite. The UTRblast utility allows database searches against fully annotated UTRdb entries.

CONCLUSIONS AND PERSPECTIVES

The important role that UTRs of eukaryotic mRNAs may play in gene regulation and expression is now widely recognized. Indeed, experimental studies have demonstrated that sequence motifs located in the UTRs are involved in crucial biological functions.

The huge amount of functionally equivalent sequences stored in UTRdb now makes possible the study of their structural and compositional features and the application of statistical methods for the identification of significant signals. Previous cleaning-up of databases is however necessary to avoid artefacts caused by redundant sequences. Even if statistical significance does not necessarily mean biological significance, it may provide useful indication for further experimental work, such as site-directed mutagenesis.

UTRdb will be updated with the new EMBL database releases and UTRsite will be continuously updated by adding new entries describing functional patterns whose biological role has been experimentally demonstrated.