Limits...
pY RNA1-s2: a highly retina-enriched small RNA that selectively binds to Matrin 3 (Matr3).

Yamazaki F, Kim HH, Lau P, Hwang CK, Iuvone PM, Klein D, Clokie SJ - PLoS ONE (2014)

Bottom Line: In contrast, pY RNA1-s1 does not bind these proteins.Hypothetically, pY RNA1-S2 might act to modulate cellular function through this molecular mechanism.The retinal enrichment of pY RNA1-s2 provides reason to suspect that the pY RNA1-s2/Matr3 interaction could play a role in vision.

View Article: PubMed Central - PubMed

Affiliation: Section on Neuroendocrinology, Program in Developmental Endocrinology and Genetics, The Eunice Shriver Kennedy National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
The purpose of this study was to expand our knowledge of small RNAs, which are known to function within protein complexes to modulate the transcriptional output of the cell. Here we describe two previously unrecognized, small RNAs, termed pY RNA1-s1 and pY RNA1-s2 (processed Y RNA1-stem -1 and -2), thereby expanding the list of known small RNAs. pY RNA1-s1 and pY RNA1-s2 were discovered by RNA sequencing and found to be 20-fold more abundant in the retina than in 14 other rat tissues. Retinal expression of pY RNAs is highly conserved, including expression in the human retina, and occurs in all retinal cell layers. Mass spectrometric analysis of pY RNA1-S2 binding proteins in retina indicates that pY RNA1-s2 selectively binds the nuclear matrix protein Matrin 3 (Matr3) and to a lesser degree to hnrpul1 (heterogeneous nuclear ribonucleoprotein U-like protein). In contrast, pY RNA1-s1 does not bind these proteins. Accordingly, the molecular mechanism of action of pY RNA1-s2 is likely be through an action involving Matr3; this 95 kDa protein has two RNA recognition motifs (RRMs) and is implicated in transcription and RNA-editing. The high affinity binding of pY RNA1-s2 to Matr3 is strongly dependent on the sequence of the RNA and both RRMs of Matr3. Related studies also indicate that elements outside of the RRM region contribute to binding specificity and that phosphorylation enhances pY RNA-s2/Matr3 binding. These observations are of significance because they reveal that a previously unrecognized small RNA, pY RNA1-s2, binds selectively to Matr3. Hypothetically, pY RNA1-S2 might act to modulate cellular function through this molecular mechanism. The retinal enrichment of pY RNA1-s2 provides reason to suspect that the pY RNA1-s2/Matr3 interaction could play a role in vision.

Show MeSH

Related in: MedlinePlus

pY RNA1-s2 selectively binds to Matr3.A. Schematic depicting the two regions of Y RNA used in a binding assay with a Maltose Binding Protein fusion with Matr3 (MBP-Matr3). Biotin groups, indicated with italic B, were chemically attached to the 3′ end of each oligo. pY RNA1-s1 is indicated with black circles and pY RNA1-s2 with grey circles. B. the RNA oligonucleotides pY RNA1-s1, pY RNA1-s2 or an equimolar mix of both oligos (pY RNA1-s1+s2) were coupled to streptavidin beads, incubated with MBP-Matr3 and then washed extensively. A lysate prepared from six rat retinas was subsequently incubated with the beads, which were then washed; the proteins associated with the beads were recovered into sample buffer, resolved by SDS-PAGE and detected by Coomassie staining (details are available in the “Materials and Methods”).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3928194&req=5

pone-0088217-g007: pY RNA1-s2 selectively binds to Matr3.A. Schematic depicting the two regions of Y RNA used in a binding assay with a Maltose Binding Protein fusion with Matr3 (MBP-Matr3). Biotin groups, indicated with italic B, were chemically attached to the 3′ end of each oligo. pY RNA1-s1 is indicated with black circles and pY RNA1-s2 with grey circles. B. the RNA oligonucleotides pY RNA1-s1, pY RNA1-s2 or an equimolar mix of both oligos (pY RNA1-s1+s2) were coupled to streptavidin beads, incubated with MBP-Matr3 and then washed extensively. A lysate prepared from six rat retinas was subsequently incubated with the beads, which were then washed; the proteins associated with the beads were recovered into sample buffer, resolved by SDS-PAGE and detected by Coomassie staining (details are available in the “Materials and Methods”).

Mentions: pY RNA1-s1 contains an imperfect antisense nucleotide sequence with pY RNA-s2, reflecting the base pairing required for formation of the stem in Y RNA1. We therefore investigated the longer pY RNA1-s1 using the same binding assay approach described in Figure 6, with the exception that a maltose binding protein fusion of Matr3 was used (MBP-Matr3; Figure 7B). The biotin-tagged oligos were used for the binding assay (Figure 7A). In contrast to the strong interaction of pY RNA1-s2 and Matr3, there was no interaction with pY RNA1-s1 and Matr3 (Figure 7B, lane 4). To investigate the possibility of Matr3 binding double stranded pY RNA1 RNA oligonucleotides, pY RNA1-s1 and pY RNA1-s2 were mixed in equimolar ratios, heated and cooled to anneal the oligos, then incubated with MBP-Matr3 as above. The annealed oligos show reduced binding to Matr3 (Figure 7B, lane 5) relative to that of pY RNA1-s2 alone.


pY RNA1-s2: a highly retina-enriched small RNA that selectively binds to Matrin 3 (Matr3).

Yamazaki F, Kim HH, Lau P, Hwang CK, Iuvone PM, Klein D, Clokie SJ - PLoS ONE (2014)

pY RNA1-s2 selectively binds to Matr3.A. Schematic depicting the two regions of Y RNA used in a binding assay with a Maltose Binding Protein fusion with Matr3 (MBP-Matr3). Biotin groups, indicated with italic B, were chemically attached to the 3′ end of each oligo. pY RNA1-s1 is indicated with black circles and pY RNA1-s2 with grey circles. B. the RNA oligonucleotides pY RNA1-s1, pY RNA1-s2 or an equimolar mix of both oligos (pY RNA1-s1+s2) were coupled to streptavidin beads, incubated with MBP-Matr3 and then washed extensively. A lysate prepared from six rat retinas was subsequently incubated with the beads, which were then washed; the proteins associated with the beads were recovered into sample buffer, resolved by SDS-PAGE and detected by Coomassie staining (details are available in the “Materials and Methods”).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3928194&req=5

pone-0088217-g007: pY RNA1-s2 selectively binds to Matr3.A. Schematic depicting the two regions of Y RNA used in a binding assay with a Maltose Binding Protein fusion with Matr3 (MBP-Matr3). Biotin groups, indicated with italic B, were chemically attached to the 3′ end of each oligo. pY RNA1-s1 is indicated with black circles and pY RNA1-s2 with grey circles. B. the RNA oligonucleotides pY RNA1-s1, pY RNA1-s2 or an equimolar mix of both oligos (pY RNA1-s1+s2) were coupled to streptavidin beads, incubated with MBP-Matr3 and then washed extensively. A lysate prepared from six rat retinas was subsequently incubated with the beads, which were then washed; the proteins associated with the beads were recovered into sample buffer, resolved by SDS-PAGE and detected by Coomassie staining (details are available in the “Materials and Methods”).
Mentions: pY RNA1-s1 contains an imperfect antisense nucleotide sequence with pY RNA-s2, reflecting the base pairing required for formation of the stem in Y RNA1. We therefore investigated the longer pY RNA1-s1 using the same binding assay approach described in Figure 6, with the exception that a maltose binding protein fusion of Matr3 was used (MBP-Matr3; Figure 7B). The biotin-tagged oligos were used for the binding assay (Figure 7A). In contrast to the strong interaction of pY RNA1-s2 and Matr3, there was no interaction with pY RNA1-s1 and Matr3 (Figure 7B, lane 4). To investigate the possibility of Matr3 binding double stranded pY RNA1 RNA oligonucleotides, pY RNA1-s1 and pY RNA1-s2 were mixed in equimolar ratios, heated and cooled to anneal the oligos, then incubated with MBP-Matr3 as above. The annealed oligos show reduced binding to Matr3 (Figure 7B, lane 5) relative to that of pY RNA1-s2 alone.

Bottom Line: In contrast, pY RNA1-s1 does not bind these proteins.Hypothetically, pY RNA1-S2 might act to modulate cellular function through this molecular mechanism.The retinal enrichment of pY RNA1-s2 provides reason to suspect that the pY RNA1-s2/Matr3 interaction could play a role in vision.

View Article: PubMed Central - PubMed

Affiliation: Section on Neuroendocrinology, Program in Developmental Endocrinology and Genetics, The Eunice Shriver Kennedy National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
The purpose of this study was to expand our knowledge of small RNAs, which are known to function within protein complexes to modulate the transcriptional output of the cell. Here we describe two previously unrecognized, small RNAs, termed pY RNA1-s1 and pY RNA1-s2 (processed Y RNA1-stem -1 and -2), thereby expanding the list of known small RNAs. pY RNA1-s1 and pY RNA1-s2 were discovered by RNA sequencing and found to be 20-fold more abundant in the retina than in 14 other rat tissues. Retinal expression of pY RNAs is highly conserved, including expression in the human retina, and occurs in all retinal cell layers. Mass spectrometric analysis of pY RNA1-S2 binding proteins in retina indicates that pY RNA1-s2 selectively binds the nuclear matrix protein Matrin 3 (Matr3) and to a lesser degree to hnrpul1 (heterogeneous nuclear ribonucleoprotein U-like protein). In contrast, pY RNA1-s1 does not bind these proteins. Accordingly, the molecular mechanism of action of pY RNA1-s2 is likely be through an action involving Matr3; this 95 kDa protein has two RNA recognition motifs (RRMs) and is implicated in transcription and RNA-editing. The high affinity binding of pY RNA1-s2 to Matr3 is strongly dependent on the sequence of the RNA and both RRMs of Matr3. Related studies also indicate that elements outside of the RRM region contribute to binding specificity and that phosphorylation enhances pY RNA-s2/Matr3 binding. These observations are of significance because they reveal that a previously unrecognized small RNA, pY RNA1-s2, binds selectively to Matr3. Hypothetically, pY RNA1-S2 might act to modulate cellular function through this molecular mechanism. The retinal enrichment of pY RNA1-s2 provides reason to suspect that the pY RNA1-s2/Matr3 interaction could play a role in vision.

Show MeSH
Related in: MedlinePlus