{"id":782,"date":"2023-03-16T13:49:24","date_gmt":"2023-03-16T06:49:24","guid":{"rendered":"https:\/\/conf.icgbio.ru\/bgrs98\/?page_id=782"},"modified":"2023-09-04T12:36:40","modified_gmt":"2023-09-04T05:36:40","slug":"059_structural-and-compositional-features-of-5-untranslated-regions-of-higher-plant-mrnas","status":"publish","type":"page","link":"https:\/\/conf.icgbio.ru\/bgrs98\/abstracts\/abstract-list\/059_structural-and-compositional-features-of-5-untranslated-regions-of-higher-plant-mrnas\/","title":{"rendered":"STRUCTURAL AND COMPOSITIONAL FEATURES OF 5\u2019 UNTRANSLATED REGIONS OF HIGHER PLANT MRNAS"},"content":{"rendered":"

KOCHETOV A.V.<\/a>+<\/sup>,\u00a0PILUGIN M.V.<\/a>,\u00a0KOLPAKOV F.A.<\/a>,\u00a0BABENKO V.N.<\/a>,\u00a0KVASHNINA E.V.<\/a>,\u00a0SHUMNY V.K.<\/a><\/p>\n

Institute of Cytology and Genetics of SD RAS, pr. Lavrentieva, 10, Novosibirsk, 630090 Russia,\u00a0ak@bionet.nsc.ru<\/a><\/p>\n

+<\/sup>Corresponding author<\/p>\n

Keywords<\/a>: structural and compositional features, 5′-untraslated regions, computer analysis, considerable asymmetry<\/p>\n

 <\/p>\n

Abstract<\/b><\/p>\n

Computer analysis of the 5\u2019untranslated regions (5\u2019UTRs) of plant mRNA sequences is reported. In comparison with 3\u2019UTRs, the nucleotide composition of 5\u2019UTRs is characterized by a considerable asymmetry in the content of the complementary nucleotides (G\/C and A\/U) and a strong decrease in the frequency of AUG triplet. It has been also shown that mRNA 5\u2019UTRs of dicot and monocot genes differ in a number of characteristics (length, nucleotide content, and context of translational start codon). Statistically, leaders of dicot mRNAs are A+U-rich sequences, and most of them (77%) vary in length between 11 and 120 nt; whereas 5\u2019UTRs of monocot mRNAs are C-rich, and their lengths are mainly distributed between 40 and 120 nt (68% in analyzed sample). Consensus sequences of translational start contexts are aaaaaaaaaAaaAUG<\/b>GCu for the dicot and ggggcggccA\/GCcAUG<\/b>GCG for the monocot mRNAs. AUG codon contexts in monocot mRNAs appeared to be considerably different from those in dicots by the occurrence of certain combinations of nucleotides in -3 and +4 positions around AUG. The doublet\u00a0G-3<\/sup>\/G+4<\/sup><\/b>\u00a0was found to be the most frequent combination in monocot mRNAs (35.3%), whereas the doublet\u00a0A-3<\/sup>\/G+4\u00a0<\/sup><\/b>was the most frequent in dicot mRNAs (35.9%).<\/p>\n

1. Introduction<\/b><\/p>\n

It is known that structural features of 5\u2019UTRs can influence the translational efficiency of eukaryotic mRNAs1<\/sup>. A scanning model2\u00a0<\/sup>is generally accepted to describe the interaction between the majority of mRNAs and ribosomes in eukaryotic cells. According to this model, 40S ribosomal subunits recognize cap at the 5\u2019mRNA end, bind to mRNA near 5\u2019end, and migrate linearly in the 3\u2019direction until they reach the first AUG codon in a favorable context. As was shown, several features of 5\u2019UTRs are important for the mRNA translation:<\/p>\n

(1) The nucleotide sequence, or context, surrounding the AUG codon.<\/i>\u00a0It is known that the efficiency of AUG codon recognition is modulated by the sequence context of the codon. For vertebrate mRNAs2<\/sup>, the most crucial positions are adenine at position -3 and a guanine at position +4 (where the A in AUG is numbered as +1), other positions seem to be less important. As for plant mRNAs, some researchers have concluded that the context of the initiation codon in plants is of minor importance3,4<\/sup>, while others have shown that it discriminates the level of initiation, similar to that in mammalian systems5,6<\/sup>. Exact role of nucleotides in different positions of the AUG context is still an open question7,8,9<\/sup>.<\/b><\/p>\n

(2)\u00a0Leader length.<\/i>\u00a0In general, longer leaders (e.g., 80 nt) result in higher translational rates in plant cells than shorter leaders (e.g., 10 nucleotides); however, mRNAs with leader sequence less than 10 nucleotides long can still be efficiently translated2,8<\/sup>.<\/p>\n

(3)\u00a0Secondary structure in the leader<\/i>. Secondary structures and AUGs within 5\u2019UTR hinder the scanning and, as a consequence, decrease translational efficiency of the mRNA. mRNAs with excessive secondary structure in their 5\u2019UTR are discriminated against by the translational machinery and inefficiently translated in both plant and animal cells1,2,6,8<\/sup>.<\/p>\n

(4)\u00a0The presence of AUGs upstream of the main initiation site<\/i>. Similar to the secondary structure, the introduction of alternative AUGs into 5\u2019UTR reduces downstream translation1,2,8<\/sup>.<\/p>\n

Only a small number of leader sequences of nuclear plant genes have been systematically compared10<\/sup>. Computer analysis of the sequences identified so far may be useful to reveal the 5\u2019UTR features influencing the mRNA translational efficiency3,9,10,11<\/sup>. In this paper we report a detailed computer analysis of the 5\u2019 leader regions of angiosperm plant mRNA sequences from the EMBL collection. The results obtained indicate significant difference between the features of angiosperm mRNA 5\u2019 and 3\u2019 untranslated sequences as well as between the dicot and monocot 5\u2019UTRs. These data may be used for predicting mRNA UTRs in newly cloned sequences and optimizing structural characteristics of foreign genes to make their expression in dicot or monocot plants more efficient.<\/p>\n

2. Materials and methods<\/b><\/p>\n

Sequence data:<\/i>\u00a0The EMBL (Release 49) database was used for this compilation. Both full-sized (i.e., when genomic DNA clone was sequenced and the transcription start site was determined in experiments; sample D) and possible incomplete (i.e., when the cDNA clone was sequenced; sample R) 5\u2019UTRs were extracted. Redundant sequences (>70%) were eliminated. Finally, database D comprised 479 sequences (including 333 of dicots and 121 of monocots) and database R comprised 3410 sequences (2475 of dicots and 748 of monocots). Databases D and R are available at\u00a0http:\/\/wwwmgs\/Dbases\/NSamples\/auto1.exe. A small database of mRNA 3\u2019UTRs of plant genes (D3\u2019) was also created. Trailer sequences were extracted from the same EMBL entries that were taken for the full-sized 5\u2019UTR database (D). The total number of mRNA 3\u2019UTRs analyzed was 315 (including 217 sequences of dicots; 82, of monocots).<\/p>\n

Sequence analysis:\u00a0<\/i>To compare the general characteristics (distributions of lengths, nucleotide content) of mRNA UTRs of different taxa (dicots and monocots), we used the Kolmogorov-Smirnov test (K.-S. test). The 50\/75 consensus rule12<\/sup>\u00a0was used to describe the AUG context consensus sequence.<\/p>\n

3. Results and discussion<\/b><\/p>\n

3.1. Length of 5\u2019UTRs<\/i><\/b><\/p>\n

Average lengths of the 5\u2019UTRs is 98 nt for dicot mRNAs and 113 nt for monocot mRNAs. The distributions of the leader lengths of the dicot and monocot mRNAs appeared significantly different (P<0.05 according to K.-S. test). The length of the most of 5\u2019UTRs of dicot mRNAs (77%) varied between 11 and 120 nt. In the case of monocots, the lengths of the 68% of the 5\u2019UTRs varied from 40 to 120 nt.<\/p>\n

3.2. Nucleotide content<\/b><\/i><\/p>\n

Nucleotide content of the 5\u2019UTRs of dicot and monocot mRNAs is reported in Table 1.<\/p>\n

 <\/p>\n

Table 1<\/p>\n

Average nucleotide content in the untranslated sequences of mRNAs of dicot (Dc) and monocot (Mc) mRNAs (samples R and D3\u2019)<\/p>\n\n\n\n\n\n\n\n\n\n\n
<\/td>\n <\/p>\n

5\u2019UTRs<\/i><\/p>\n<\/td>\n

 <\/p>\n

3\u2019UTRs<\/i><\/p>\n<\/td>\n<\/tr>\n

 <\/p>\n

Taxon<\/b><\/p>\n<\/td>\n

 <\/p>\n

Dc<\/b><\/p>\n<\/td>\n

 <\/p>\n

Mc<\/b><\/p>\n<\/td>\n

 <\/p>\n

Dc<\/b><\/p>\n<\/td>\n

 <\/p>\n

Mc<\/b><\/p>\n<\/td>\n<\/tr>\n

A<\/b><\/td>\n\n

32.1<\/p>\n<\/td>\n

\n

23.4<\/p>\n<\/td>\n

\n

29.9<\/p>\n<\/td>\n

\n

26.1<\/p>\n<\/td>\n<\/tr>\n

G<\/b><\/td>\n\n

15.7<\/p>\n<\/td>\n

\n

23.5<\/p>\n<\/td>\n

\n

17.0<\/p>\n<\/td>\n

\n

22.2<\/p>\n<\/td>\n<\/tr>\n

C<\/b><\/td>\n\n

22.7<\/p>\n<\/td>\n

\n

33.2<\/p>\n<\/td>\n

\n

14.4<\/p>\n<\/td>\n

\n

19.9<\/p>\n<\/td>\n<\/tr>\n

U<\/b><\/td>\n\n

29.5<\/p>\n<\/td>\n

\n

20.0<\/p>\n<\/td>\n

\n

38.5<\/p>\n<\/td>\n

\n

31.8<\/p>\n<\/td>\n<\/tr>\n

A+U<\/b><\/td>\n\n

61.6<\/p>\n<\/td>\n

\n

43.3<\/p>\n<\/td>\n

\n

68.5<\/p>\n<\/td>\n

\n

57.9<\/p>\n<\/td>\n<\/tr>\n

A+U\/G+C<\/b><\/td>\n\n

1.80<\/p>\n<\/td>\n

\n

0.85<\/p>\n<\/td>\n

\n

2.24<\/p>\n<\/td>\n

\n

1.42<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

According to these results, there is a considerable difference between the nucleotide content in dicot and monocot mRNA leader sequences: 5\u2019UTRs of dicot mRNAs are A+U-rich sequences, whereas those of monocot mRNAs are mainly C-rich. Thus, the A+U-richness of plant mRNA leaders showed previously10<\/sup>\u00a0is a characteristic feature of dicot 5\u2019UTRs only.<\/p>\n

Analysis of the distributions of the nucleotide content of mRNA untranslated sequences showed that 5\u2019UTRs of dicot and monocot mRNAs are significantly different (P<0.001 for each of the four nucleotides, according to K.-S. test).\u00a0It was demonstrated that the majority of 5\u2019UTRs of dicot mRNAs contain 30-50% of A, up to 30% of G, 10-30% of C, and 20-40% of U; whereas most of 5\u2019UTRs of monocot mRNAs contain 10-40% of A and G, 20-50% of C, and 10-30% of U.<\/span><\/p>\n

The nucleotide content of mRNA 3\u2019UTRs was also determined. It was found that 5\u2019 and 3\u2019 untranslated regions of angiosperm mRNAs are characterized by a strong difference in the content of\u00a0pyrimidines,<\/b>\u00a0whereas the concentrations of\u00a0purines<\/b>\u00a0are close. The concentration of U is higher in 3\u2019UTRs, whereas the concentration of C is higher in the 5\u2019UTRs of both dicot and monocot genes. Comparison of the distributions of nucleotide contents in 3\u2019UTRs of dicot and monocot mRNAs (by K.-S. test) showed that they are significantly different for each of the four nucleotides (P<0.001).\u00a0It was shown that the most part of 3\u2019UTRs of dicot mRNAs contain 20-40% of A, 10-20% of G and C, and 30-50% of U; whereas most of 3\u2019UTRs of monocot mRNAs contain 20-30% of A and G, 10-30% of C, and 30-40% of U.<\/span><\/p>\n

It is likely that the difference in nucleotide compositions of 5\u2019 and 3\u2019 untranslated sequences is essential for their specific functions. As to the difference between the distributions of nucleotide content between 5\u2019UTRs of dicot and 5\u2019UTRs of monocot mRNAs, it may result from the difference in their genome organization13<\/sup>.<\/p>\n

The G+C richness of a 5\u2019UTR sequence is considered indicative of the stability of the potential secondary structure, whose formation may hinder the movement of the 40S ribosomal subunit along mRNA during the scanning process2<\/sup>. We analyzed the plant mRNA untranslated sequences for the ratios of the frequencies of the complementary nucleotides involved in the formation of the secondary structure (G\/C and A\/U). It was found that the G and C concentrations were close (0.8<G\/C (A\/U)<1.2) only in 21.7% of the leaders in the sample of the dicot mRNAs, whereas they constituted 36.8% in the corresponding sample of the 3\u2019UTRs. Similarly, the A and U concentrations were close in 27.5% of the 5\u2019UTRs of the dicot mRNAs and in 41.5% of the 3\u2019UTRs. Similar patterns were found for the G\/C and A\/U ratios in the untranslated sequences of monocot mRNAs. A high asymmetry in the content of the complementary nucleotides found in 5\u2019UTRs decreases the stability of the potential secondary structure. It may be related to the specific roles of these types of sequences: prevention of the formation of the stable secondary structure in mRNA leaders is of importance to provide the conditions for high mRNA translation efficiency.<\/p>\n

3.3. AUG codon context<\/b><\/i><\/p>\n

To analyze the context of translational initiation site, mRNA fragments (from -12 nt to +6 nt around AUG) were aligned (data not shown). It was found that the consensus sequences of translational start contexts are aaaaaaaaaAaaAUG<\/b>GCu for the dicot and ggggcggccA\/GCcAUG<\/b>GCG for the monocot mRNAs (according to 50\/75 consensus rule12<\/sup>). The frequencies of G and A in -3 position upstream of AUG in monocot mRNAs appeared to be very close (44.3% and 33.6%, respectively). It is likely that their influence on the context \u201cstrength\u201d in monocots is equal. It was suggested2,14<\/sup>\u00a0that nucleotides in -3 and +4 positions make a major contributions to the translation from the AUG codon. A in -3 and G in +4 positions are optimal. We analyzed the frequencies of translational start sites surrounded by several combinations of nucleotides in these positions in the dicot and monocot mRNA samples (Table 2).<\/p>\n

 <\/p>\n

Table 2. Frequencies (%) of the dicot and monocot mRNAs (sample R) containing certain combinations of nucleotides in positions -3 and +4 around the start codon*<\/p>\n\n\n\n\n\n\n\n\n\n
AUG type (Nu-3<\/sup>;Nu+4<\/sup>)<\/td>\n\n

Dicot mRNA<\/p>\n<\/td>\n

\n

Monocot mRNAs<\/p>\n<\/td>\n<\/tr>\n

1 (A; G)<\/td>\n\n

35.9<\/p>\n<\/td>\n

 <\/p>\n

19.8<\/i><\/p>\n<\/td>\n<\/tr>\n

2 (G; G)<\/td>\n\n

16.5<\/p>\n<\/td>\n

 <\/p>\n

35.3<\/b><\/p>\n<\/td>\n<\/tr>\n

3 (Py; G)<\/td>\n <\/p>\n

14.9<\/b><\/p>\n<\/td>\n

\n

15.1<\/p>\n<\/td>\n<\/tr>\n

4 (A; not G)<\/td>\n <\/p>\n

20.8<\/b><\/p>\n<\/td>\n

 <\/p>\n

16.8<\/b><\/p>\n<\/td>\n<\/tr>\n

5 (G; not G)<\/td>\n\n

7.1<\/p>\n<\/td>\n

 <\/p>\n

9.0<\/i><\/p>\n<\/td>\n<\/tr>\n

6 (Py; not G)<\/td>\n <\/p>\n

4.8<\/i><\/p>\n<\/td>\n

 <\/p>\n

4.0<\/i><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

*The frequencies that are significantly higher than the expected** are boldface; those lower, italicized (P<0.05, one degree of freedom; \"\"\u00a0values are not shown).<\/p>\n

**Expected frequencies were calculated as P1*P2 where P1 and P2 are mean concentrations of nucleotides in positions -3 and +4 (data not shown).<\/p>\n

 <\/p>\n

The frequencies of the AUG codon containing guanines in positions -3 and +4 appeared to be considerably different in the dicot (16.5%) and monocot (35.3%) mRNA samples. It is likely that the G-3<\/sup>\/G+4\u00a0<\/sup>combination provides at least equal contribution to the AUG translational activity as the A-3<\/sup>\/G+4<\/sup>\u00a0does in monocots. A higher frequency of combination A-3<\/sup>\/ not G+4\u00a0<\/sup>over the G-3<\/sup>\/not G+4\u00a0<\/sup>may result from the dependence of G-3<\/sup>\u00a0activity upon the presence of guanine in position +4 for both dicot and monocot mRNAs.<\/p>\n

3.4. The presence of AUG codons in 5\u2019UTRs<\/b><\/i><\/p>\n

It was shown that upstream AUG codons decrease the mRNA translational efficiency in eukaryotic cells2,8<\/sup>. We determined the extent of deviation of the observed AUG frequencies in 5\u2019UTRs of dicot and monocot mRNAs normalized to the corresponding 5\u2019UTR lengths from those expected (the expected AUG frequencies were calculated as Paug=Pa*Pu*Pg, where Px is an average frequency of the nucleotide X in 5\u2019UTRs of dicot or monocot mRNAs). It appeared that the ratios of Obs to Exp AUG frequencies in 5\u2019UTRs of dicot and monocot mRNAs (sample D) were considerably lower (0.371 and 0.377, respectively) than for the 3\u2019UTRs (1.22 and 1.28, respectively). We have found that the frequencies of AUG-containing 5\u2019UTR are relatively high (13-19.5% in the samples D and R) in both dicot and monocot mRNAs and significantly exceed those reported for vertebrate mRNAs2<\/sup>. It seems likely that the negative influence of upstream AUGs in plant mRNAs8<\/sup>\u00a0may be compensated by still unknown mechanisms allowing to discriminate between the true and false starts15<\/sup>.<\/p>\n

Acknowledgments<\/b><\/p>\n

A.K. was supported by the SD RAS grant for young scientists. V.Sh. benefited from the RFBR Program of Support of Scientific Schools (Russia).<\/p>\n

References<\/b><\/p>\n

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    KOCHETOV A.V.+,\u00a0PILUGIN M.V.,\u00a0KOLPAKOV F.A.,\u00a0BABENKO V.N.,\u00a0KVASHNINA E.V.,\u00a0SHUMNY V.K. Institute of Cytology and Genetics of SD RAS, pr. Lavrentieva, 10, Novosibirsk, 630090 Russia,\u00a0ak@bionet.nsc.ru +Corresponding author Keywords: structural and compositional features, 5′-untraslated regions, computer analysis, considerable asymmetry   Abstract Computer analysis of the … Continue reading →<\/span><\/a><\/p>\n","protected":false},"author":13,"featured_media":0,"parent":97,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":[],"_links":{"self":[{"href":"https:\/\/conf.icgbio.ru\/bgrs98\/wp-json\/wp\/v2\/pages\/782"}],"collection":[{"href":"https:\/\/conf.icgbio.ru\/bgrs98\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/conf.icgbio.ru\/bgrs98\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/conf.icgbio.ru\/bgrs98\/wp-json\/wp\/v2\/users\/13"}],"replies":[{"embeddable":true,"href":"https:\/\/conf.icgbio.ru\/bgrs98\/wp-json\/wp\/v2\/comments?post=782"}],"version-history":[{"count":5,"href":"https:\/\/conf.icgbio.ru\/bgrs98\/wp-json\/wp\/v2\/pages\/782\/revisions"}],"predecessor-version":[{"id":1495,"href":"https:\/\/conf.icgbio.ru\/bgrs98\/wp-json\/wp\/v2\/pages\/782\/revisions\/1495"}],"up":[{"embeddable":true,"href":"https:\/\/conf.icgbio.ru\/bgrs98\/wp-json\/wp\/v2\/pages\/97"}],"wp:attachment":[{"href":"https:\/\/conf.icgbio.ru\/bgrs98\/wp-json\/wp\/v2\/media?parent=782"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}