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Differential pre-mRNA Splicing Alters the Transcript Diversity of Helitrons Between the Maize Inbred Lines.

Lynch BT, Patrick TL, Moreno JJ, Siebert AE, Klusman KM, Shodja DN, Hannah LC, Lal SK - G3 (Bethesda) (2015)

Bottom Line: The comparison of Helitron sequences identified unique polymorphisms in inbred B73, which potentially give rise to the alternatively spliced sites utilized by transcript isoforms.Some alterations in splicing, however, do not have obvious explanations.These observations not only add another level to the creation of transcript diversity by Helitrons among inbred lines but also provide novel insights into the cis-acting elements governing splice-site selection during pre-mRNA processing.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Oakland University, Rochester, Minnesota 48309-4401.

No MeSH data available.


Alternatively spliced transcript isoforms of Helitron Hel1-332 display inbred-specific expression. (A) Gene structure of the six transcript isoforms I−VI of Hel1-332 in inbred line B73 (Barbaglia et al. 2012). The transcript isoforms, specific to B73 and not detected in other inbred lines, are highlighted in green. The noncanonical splice sites are shown in blue, and asterisks mark the retained introns. (B) Junction sequence alignment between the inbred lines of the splice sites marked by the lower case Roman numerals in (A).
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fig3: Alternatively spliced transcript isoforms of Helitron Hel1-332 display inbred-specific expression. (A) Gene structure of the six transcript isoforms I−VI of Hel1-332 in inbred line B73 (Barbaglia et al. 2012). The transcript isoforms, specific to B73 and not detected in other inbred lines, are highlighted in green. The noncanonical splice sites are shown in blue, and asterisks mark the retained introns. (B) Junction sequence alignment between the inbred lines of the splice sites marked by the lower case Roman numerals in (A).

Mentions: In the same manner, to decipher the molecular basis of RT-PCR differences observed between these maize inbred lines, we cloned and sequenced a ∼3.9-kb fragment of Helitron Hel1-332 PCR amplified from the genomic DNA isolated from inbred lines HP301, OH7B and Tzi8. Figure S2 displays the multiple alignment of the Hel1-332 sequence from inbred lines B73, HP301, OH7B, and Tzi8. As shown, a high degree of similarity is shared between the Hel1-332 sequences of the four inbred lines even though regions of polymorphism, including indels, span the entire length of the element. The sequence of inbred line HP301 displayed highest similarity of >99% with B73, whereas OH7B shared 97% and Tzi8 had 94% similarity with B73. The alternative splicing produces six isoforms of Hel1-332 transcript in inbred line B73 (Barbaglia et al. 2012). In addition, we sequenced the two products amplified from the inbred lines HP301 and OH7B and a single product from inbred Tzi8. We performed splice alignment of the resultant products with their corresponding Helitron in different inbred lines to determine the gene structure and position of the intron/exon junctions. The gene structure of a single product of Hel1-332 amplified from inbred Tzi8 is identical to transcript I of inbred B73 (Figure 3). Similarly, the two products amplified from HP301 displayed identical gene structure to transcripts I and II of inbred B73, whereas the two transcripts of OH7B were identical to transcripts II and V of B73. The transcripts III, IV, and VI were unique to B73 and were absent in other inbred lines. The majority of splicing events that give birth to various transcript isoforms are concentrated in the 5′ end of the Helitron, spanning exon 1 and intron 1 of transcript I (Figure 3). The exception is the retention of an unspliced intron 5 of transcript I in transcript VI spanning the 3′ end of the Helitron. The alternative splicing events that use donor or acceptor sites different from transcript I are displayed in Figure 2, A and B. As shown, the use of a noncanonical donor site within intron 1 and an acceptor site of exon 2 produces transcript II of B73. The flanking A at −10 position of the donor site in B73 was replaced with G in OH7B and Tzi8. Similarly, an alternative donor site within exon 1, an acceptor site of exon 2, and retention of an entire intron 1 give rise to B73 transcripts III and IV, respectively. As displayed in Figure 2B, no polymorphism was detected flanking the splice-site junction between the inbred lines of these splicing events. The use of several donor and acceptor sites creates two exons within intron 1 of B73 transcript V. There exists no polymorphism between the inbred lines flanking the splice junction of the first intron of this transcript. However, a flanking A at −2 position of the acceptor site of the second intron region in B73 was replaced by G in inbred Tzi8. In transcript vi, a G in inbred Tzi8 replaced T at −3 position of the acceptor site of the third intron region in B73. Similarly, T at +9 position of the acceptor site was replaced by C in inbred HP301. The retention of a complete intron 5 of B73 transcript isoform I produced transcript isoform VI. In transcript VI, the A at −15 position and T at −2 position in inbred B73 were both replaced by C in OH7B and Tzi8. The other splicing events common to all the six transcript isoforms of B73 did not display any junction site flanking polymorphism between the inbred lines.


Differential pre-mRNA Splicing Alters the Transcript Diversity of Helitrons Between the Maize Inbred Lines.

Lynch BT, Patrick TL, Moreno JJ, Siebert AE, Klusman KM, Shodja DN, Hannah LC, Lal SK - G3 (Bethesda) (2015)

Alternatively spliced transcript isoforms of Helitron Hel1-332 display inbred-specific expression. (A) Gene structure of the six transcript isoforms I−VI of Hel1-332 in inbred line B73 (Barbaglia et al. 2012). The transcript isoforms, specific to B73 and not detected in other inbred lines, are highlighted in green. The noncanonical splice sites are shown in blue, and asterisks mark the retained introns. (B) Junction sequence alignment between the inbred lines of the splice sites marked by the lower case Roman numerals in (A).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: Alternatively spliced transcript isoforms of Helitron Hel1-332 display inbred-specific expression. (A) Gene structure of the six transcript isoforms I−VI of Hel1-332 in inbred line B73 (Barbaglia et al. 2012). The transcript isoforms, specific to B73 and not detected in other inbred lines, are highlighted in green. The noncanonical splice sites are shown in blue, and asterisks mark the retained introns. (B) Junction sequence alignment between the inbred lines of the splice sites marked by the lower case Roman numerals in (A).
Mentions: In the same manner, to decipher the molecular basis of RT-PCR differences observed between these maize inbred lines, we cloned and sequenced a ∼3.9-kb fragment of Helitron Hel1-332 PCR amplified from the genomic DNA isolated from inbred lines HP301, OH7B and Tzi8. Figure S2 displays the multiple alignment of the Hel1-332 sequence from inbred lines B73, HP301, OH7B, and Tzi8. As shown, a high degree of similarity is shared between the Hel1-332 sequences of the four inbred lines even though regions of polymorphism, including indels, span the entire length of the element. The sequence of inbred line HP301 displayed highest similarity of >99% with B73, whereas OH7B shared 97% and Tzi8 had 94% similarity with B73. The alternative splicing produces six isoforms of Hel1-332 transcript in inbred line B73 (Barbaglia et al. 2012). In addition, we sequenced the two products amplified from the inbred lines HP301 and OH7B and a single product from inbred Tzi8. We performed splice alignment of the resultant products with their corresponding Helitron in different inbred lines to determine the gene structure and position of the intron/exon junctions. The gene structure of a single product of Hel1-332 amplified from inbred Tzi8 is identical to transcript I of inbred B73 (Figure 3). Similarly, the two products amplified from HP301 displayed identical gene structure to transcripts I and II of inbred B73, whereas the two transcripts of OH7B were identical to transcripts II and V of B73. The transcripts III, IV, and VI were unique to B73 and were absent in other inbred lines. The majority of splicing events that give birth to various transcript isoforms are concentrated in the 5′ end of the Helitron, spanning exon 1 and intron 1 of transcript I (Figure 3). The exception is the retention of an unspliced intron 5 of transcript I in transcript VI spanning the 3′ end of the Helitron. The alternative splicing events that use donor or acceptor sites different from transcript I are displayed in Figure 2, A and B. As shown, the use of a noncanonical donor site within intron 1 and an acceptor site of exon 2 produces transcript II of B73. The flanking A at −10 position of the donor site in B73 was replaced with G in OH7B and Tzi8. Similarly, an alternative donor site within exon 1, an acceptor site of exon 2, and retention of an entire intron 1 give rise to B73 transcripts III and IV, respectively. As displayed in Figure 2B, no polymorphism was detected flanking the splice-site junction between the inbred lines of these splicing events. The use of several donor and acceptor sites creates two exons within intron 1 of B73 transcript V. There exists no polymorphism between the inbred lines flanking the splice junction of the first intron of this transcript. However, a flanking A at −2 position of the acceptor site of the second intron region in B73 was replaced by G in inbred Tzi8. In transcript vi, a G in inbred Tzi8 replaced T at −3 position of the acceptor site of the third intron region in B73. Similarly, T at +9 position of the acceptor site was replaced by C in inbred HP301. The retention of a complete intron 5 of B73 transcript isoform I produced transcript isoform VI. In transcript VI, the A at −15 position and T at −2 position in inbred B73 were both replaced by C in OH7B and Tzi8. The other splicing events common to all the six transcript isoforms of B73 did not display any junction site flanking polymorphism between the inbred lines.

Bottom Line: The comparison of Helitron sequences identified unique polymorphisms in inbred B73, which potentially give rise to the alternatively spliced sites utilized by transcript isoforms.Some alterations in splicing, however, do not have obvious explanations.These observations not only add another level to the creation of transcript diversity by Helitrons among inbred lines but also provide novel insights into the cis-acting elements governing splice-site selection during pre-mRNA processing.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Oakland University, Rochester, Minnesota 48309-4401.

No MeSH data available.