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Intronic Alus influence alternative splicing.

Lev-Maor G, Ram O, Kim E, Sela N, Goren A, Levanon EY, Ast G - PLoS Genet. (2008)

Bottom Line: A large fraction of Alus are found in intronic sequences, implying extensive Alu-Alu dsRNA formation in mRNA precursors.Yet, the effect of these intronic Alus on splicing of the flanking exons is largely unknown.Our results indicate the importance of intronic Alus in influencing the splicing of flanking exons, further emphasizing the role of Alus in shaping of the human transcriptome.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Molecular Genetics, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

ABSTRACT
Examination of the human transcriptome reveals higher levels of RNA editing than in any other organism tested to date. This is indicative of extensive double-stranded RNA (dsRNA) formation within the human transcriptome. Most of the editing sites are located in the primate-specific retrotransposed element called Alu. A large fraction of Alus are found in intronic sequences, implying extensive Alu-Alu dsRNA formation in mRNA precursors. Yet, the effect of these intronic Alus on splicing of the flanking exons is largely unknown. Here, we show that more Alus flank alternatively spliced exons than constitutively spliced ones; this is especially notable for those exons that have changed their mode of splicing from constitutive to alternative during human evolution. This implies that Alu insertions may change the mode of splicing of the flanking exons. Indeed, we demonstrate experimentally that two Alu elements that were inserted into an intron in opposite orientation undergo base-pairing, as evident by RNA editing, and affect the splicing patterns of a downstream exon, shifting it from constitutive to alternative. Our results indicate the importance of intronic Alus in influencing the splicing of flanking exons, further emphasizing the role of Alus in shaping of the human transcriptome.

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Distance effect of Alu elements on the alternative splicing pattern.(A) A schematic illustration of the genomic region between exons 2 and 3 of RABL5 gene. Arrows marked A and B indicate two positions where an intronic sequence was inserted. (B) An 800-nucleotide intronic sequence was inserted in site B. The 800-nucleotide sequence was gradually shortened to the size shown above each lane. The indicated wt and chimeric plasmids were transfected into human 293T cells, total RNA was collected and examined by RT-PCR analysis (lanes 1–9). Lanes 10–12 show insertions of a different sequence, containing 25 nucleotides without any known splicing regulatory sequences, into the same site. This sequence was duplicated and triplicated to generate 50- and 75-nucleotide inserts. These mutant RABL5 minigenes were examined as above. (C) Similar analysis as in panel B, except that the 800-nucleotide sequence and the shorter sequences were inserted into site A. Spliced products are shown on the right and each PCR product was confirmed by sequencing. Splicing products were separated on a 2% agarose gel. Numbers on top of the gel indicate percentage of exon inclusion as determined using ImageJ software.
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pgen-1000204-g004: Distance effect of Alu elements on the alternative splicing pattern.(A) A schematic illustration of the genomic region between exons 2 and 3 of RABL5 gene. Arrows marked A and B indicate two positions where an intronic sequence was inserted. (B) An 800-nucleotide intronic sequence was inserted in site B. The 800-nucleotide sequence was gradually shortened to the size shown above each lane. The indicated wt and chimeric plasmids were transfected into human 293T cells, total RNA was collected and examined by RT-PCR analysis (lanes 1–9). Lanes 10–12 show insertions of a different sequence, containing 25 nucleotides without any known splicing regulatory sequences, into the same site. This sequence was duplicated and triplicated to generate 50- and 75-nucleotide inserts. These mutant RABL5 minigenes were examined as above. (C) Similar analysis as in panel B, except that the 800-nucleotide sequence and the shorter sequences were inserted into site A. Spliced products are shown on the right and each PCR product was confirmed by sequencing. Splicing products were separated on a 2% agarose gel. Numbers on top of the gel indicate percentage of exon inclusion as determined using ImageJ software.

Mentions: We next set to examine if the distance between exon 3 and the intronic Alus and the distance between Alu1 and Alu2 were important factors in splicing of exon 3. Alu2 is located 24 nucleotides upstream of exon 3. We identified the putative branch site of intron 2 and inserted an 800-nucleotide non-Alu intronic sequence upstream of the branch sequence and downstream of intronic Alu1 and Alu2 (marked B in Figure 4A; see Text S3). This insertion caused a shift from alternative to constitutive inclusion of exon 3 (Figure 4B, compare lane 1 and 2). Only when this insertion was shortened to less than 68 nucleotides did we begin to detect restoration of alternative splicing of exon 3; the level of skipping was further elevated when the inserted sequence was shortened to 56 or to 44 nucleotides (Figure 4B, lanes 3–9). To rule out the possibility that the sequence that was inserted contained intronic splicing regulatory sequences, we designed a fragment of 25 nucleotides free from known splicing regulatory sequences (see Materials and Methods). We inserted this sequence into site B and also duplicated and triplicated this sequence to generate 50 and 75 nucleotides insertions. The longer is the inserted sequence, the higher is the inclusion level (Figure 4B, lanes 10–13). This indicates that the distance between the intronic Alu2 from exon 3 affects the mode of splicing.


Intronic Alus influence alternative splicing.

Lev-Maor G, Ram O, Kim E, Sela N, Goren A, Levanon EY, Ast G - PLoS Genet. (2008)

Distance effect of Alu elements on the alternative splicing pattern.(A) A schematic illustration of the genomic region between exons 2 and 3 of RABL5 gene. Arrows marked A and B indicate two positions where an intronic sequence was inserted. (B) An 800-nucleotide intronic sequence was inserted in site B. The 800-nucleotide sequence was gradually shortened to the size shown above each lane. The indicated wt and chimeric plasmids were transfected into human 293T cells, total RNA was collected and examined by RT-PCR analysis (lanes 1–9). Lanes 10–12 show insertions of a different sequence, containing 25 nucleotides without any known splicing regulatory sequences, into the same site. This sequence was duplicated and triplicated to generate 50- and 75-nucleotide inserts. These mutant RABL5 minigenes were examined as above. (C) Similar analysis as in panel B, except that the 800-nucleotide sequence and the shorter sequences were inserted into site A. Spliced products are shown on the right and each PCR product was confirmed by sequencing. Splicing products were separated on a 2% agarose gel. Numbers on top of the gel indicate percentage of exon inclusion as determined using ImageJ software.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2533698&req=5

pgen-1000204-g004: Distance effect of Alu elements on the alternative splicing pattern.(A) A schematic illustration of the genomic region between exons 2 and 3 of RABL5 gene. Arrows marked A and B indicate two positions where an intronic sequence was inserted. (B) An 800-nucleotide intronic sequence was inserted in site B. The 800-nucleotide sequence was gradually shortened to the size shown above each lane. The indicated wt and chimeric plasmids were transfected into human 293T cells, total RNA was collected and examined by RT-PCR analysis (lanes 1–9). Lanes 10–12 show insertions of a different sequence, containing 25 nucleotides without any known splicing regulatory sequences, into the same site. This sequence was duplicated and triplicated to generate 50- and 75-nucleotide inserts. These mutant RABL5 minigenes were examined as above. (C) Similar analysis as in panel B, except that the 800-nucleotide sequence and the shorter sequences were inserted into site A. Spliced products are shown on the right and each PCR product was confirmed by sequencing. Splicing products were separated on a 2% agarose gel. Numbers on top of the gel indicate percentage of exon inclusion as determined using ImageJ software.
Mentions: We next set to examine if the distance between exon 3 and the intronic Alus and the distance between Alu1 and Alu2 were important factors in splicing of exon 3. Alu2 is located 24 nucleotides upstream of exon 3. We identified the putative branch site of intron 2 and inserted an 800-nucleotide non-Alu intronic sequence upstream of the branch sequence and downstream of intronic Alu1 and Alu2 (marked B in Figure 4A; see Text S3). This insertion caused a shift from alternative to constitutive inclusion of exon 3 (Figure 4B, compare lane 1 and 2). Only when this insertion was shortened to less than 68 nucleotides did we begin to detect restoration of alternative splicing of exon 3; the level of skipping was further elevated when the inserted sequence was shortened to 56 or to 44 nucleotides (Figure 4B, lanes 3–9). To rule out the possibility that the sequence that was inserted contained intronic splicing regulatory sequences, we designed a fragment of 25 nucleotides free from known splicing regulatory sequences (see Materials and Methods). We inserted this sequence into site B and also duplicated and triplicated this sequence to generate 50 and 75 nucleotides insertions. The longer is the inserted sequence, the higher is the inclusion level (Figure 4B, lanes 10–13). This indicates that the distance between the intronic Alu2 from exon 3 affects the mode of splicing.

Bottom Line: A large fraction of Alus are found in intronic sequences, implying extensive Alu-Alu dsRNA formation in mRNA precursors.Yet, the effect of these intronic Alus on splicing of the flanking exons is largely unknown.Our results indicate the importance of intronic Alus in influencing the splicing of flanking exons, further emphasizing the role of Alus in shaping of the human transcriptome.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Molecular Genetics, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

ABSTRACT
Examination of the human transcriptome reveals higher levels of RNA editing than in any other organism tested to date. This is indicative of extensive double-stranded RNA (dsRNA) formation within the human transcriptome. Most of the editing sites are located in the primate-specific retrotransposed element called Alu. A large fraction of Alus are found in intronic sequences, implying extensive Alu-Alu dsRNA formation in mRNA precursors. Yet, the effect of these intronic Alus on splicing of the flanking exons is largely unknown. Here, we show that more Alus flank alternatively spliced exons than constitutively spliced ones; this is especially notable for those exons that have changed their mode of splicing from constitutive to alternative during human evolution. This implies that Alu insertions may change the mode of splicing of the flanking exons. Indeed, we demonstrate experimentally that two Alu elements that were inserted into an intron in opposite orientation undergo base-pairing, as evident by RNA editing, and affect the splicing patterns of a downstream exon, shifting it from constitutive to alternative. Our results indicate the importance of intronic Alus in influencing the splicing of flanking exons, further emphasizing the role of Alus in shaping of the human transcriptome.

Show MeSH