Specific sequence determinants of miR-15/107 microRNA gene group targets

Abstract

MicroRNAs (miRNAs) target mRNAs in human cells via complex mechanisms that are still incompletely understood. Using anti-Argonaute (anti-AGO) antibody co-immunoprecipitation, followed by microarray analyses and downstream bioinformatics, 'RIP-Chip' experiments enable direct analyses of miRNA targets. RIP-Chip studies (and parallel assessments of total input mRNA) were performed in cultured H4 cells after transfection with miRNAs corresponding to the miR-15/107 gene group (miR-103, miR-107, miR-16 and miR-195), and five control miRNAs. Three biological replicates were run for each condition with a total of 54 separate human Affymetrix Human Gene 1.0 ST array replicates. Computational analyses queried for determinants of miRNA:mRNA binding. The analyses support four major findings: (i) RIP-Chip studies correlated with total input mRNA profiling provides more comprehensive information than using either RIP-Chip or total mRNA profiling alone after miRNA transfections; (ii) new data confirm that miR-107 paralogs target coding sequence (CDS) of mRNA; (iii) biochemical and computational studies indicate that the 3' portion of miRNAs plays a role in guiding miR-103/7 to the CDS of targets; and (iv) there are major sequence-specific targeting differences between miRNAs in terms of CDS versus 3'-untranslated region targeting, and stable AGO association versus mRNA knockdown. Future studies should take this important miRNA-to-miRNA variability into account.

Figure 1.

Experimental design of miRNA transfection experiments followed by anti-AGO co-immunoprecipitation (anti-AGO Co-IP) and microarray profiling of mRNAs from the anti-AGO Co-IP and lysates. Downstream bioinformatics allowed comparison between the mRNA ‘targets’ seen in the RIP-Chip experiments, relative to the mRNAs ‘knocked down’ in the lysates following miRNA transfections.

Figure 2.

List of miRNAs transfected in the current study. The 5′ seed sequence (nucleotides 2–7) of the miRNAs is indicated. Note that there are three non-physiological controls—miR-107 MUT1, miR-107 MUT2 and NEG-CTRL. miR-15b* is an anti-sense control miRNA. Note that four members of the miR-15/107 gene family of miRNAs (miR-16, miR-195, miR-103 and miR-107) have similar 5′-seed sequences.

Figure 3.

Top 15 targets (according to anti-AGO co-IP experiments) for miR-107, miR-107 MUT1 (5′ portion mutated) and miR-107 MUT2 (3′ portion mutated). Note that the miR-107 and miR-107 MUT2 have the same 5′-seed sequence (GCAGCA which is complementary to UGCUGC on the mRNA), but miR-107 MUT1 has a different 5′ seed (CGUCGC). The number of those seed sequences in the 5′-UTR, CDS/open reading frame and 3′-UTR for each of the targets is shown. Note that for the top 15 miR-107 targets, miR-107 seed sequences tend to be found in the CDS. For miR-107 MUT1, which has a completely different 5′-seed sequence, that seed sequence is found in the CDS of 8/15 top 15 targets (versus 0/15 for top 15 miR-107 targets). In contrast, miR-107 MUT2 has the same 5′-seed sequence as miR-107, but there is a remarkable tendency among the top 15 targets of miR-107 MUT2 to include mRNAs with the miR-107 seed sequence in the 3′-UTR instead of the CDS. These data appear to indicate that the 3′ portion of miR-107 may be directing the miRNA to target CDS rather than the 3′-UTR.

Figure 4.

miR-103 seed sequences are highly enriched in open reading frame (CDS) of targets. As for miR-103, the strongest miR-103 targets are those that are enriched for the complementary 5′-seed sequence in the CDS. These data correlate closely with results previously reported (22). To visualize the correlation between enrichment in the Anti-AGO Co-IP and the seed sequence densities in the 5′-UTR, CDS and 3′-UTR, we first arranged the mRNAs according to the enrichment in the anti-AGO Co-IP (A). Here one sees the top 3000 mRNAs in the order of the average expression levels detected on the microarray using RNA from the Anti-AGO Co-IP RNA, relative to the transfection using the NEG-CTRL miRNA (N = 3 each). This same order of mRNAs was used for the chart in B, which shows the miR-103 5′ complementary seed densities in 5′-UTR, CDS and 3′-UTR. The mRNAs are binned with each data point representing the average seed densities per 1000 nt for 50 different mRNAs. Note that the most-enriched 50 mRNAs (red arrow) have by far the highest miR-103 seed densities in the CDS. However, there remains some degree of enrichment for miR-103 5′-seed sequences even among the less-enriched mRNAs (orange arrow). In contrast, when the mRNAs are sorted randomly (C, where mRNAs are sorted according to the enrichment following miR-15b* transfection), there is lower miR-103 seed sequences in the 5′-UTR, CDS and 3′-UTR alike.

Figure 5.

Enrichment of 5′ miRNA seed sequences for miR-103 in 5′-UTR, CDS or 3′-UTR does not correlate with decreased mRNA in the lysate. Across all the evaluated mRNA, there is no evidence that for miR-103 (or miR-107, data not shown) that mRNAs ‘knocked down’ in the lysate after miRNA transfection correlate to those mRNAs with 5′-seed sequences enriched in 5′-UTR, CDS or 3′-UTR. In this case, the mRNAs are ordered (x-axis) based on the degree of decrease in the lysate following miR-103 transfection.

Figure 6.

Enrichment of 5′ miRNA seed sequences for miR-320 in the 3′-UTR of targets correlates with decreased mRNA levels for those targets in the cell lysates. For miR-320, unlike miR-103 or miR-107, there is robust evidence that the seed sequences in the 3′-UTR (as opposed to in the CDS) play a strong role in determining the miRNA/mRNA targeting. Also unlike miR-103 and miR-107, miR-320 transfections appear to result in target knockdown. Note that the seed sequences that are disproportionately found in the Anti-AGO Co-IP enriched miRNAs (A) and also the mRNAs knocked down in the lysate after miR-320 transfection (B), correspond to seed sequences in the 3′-UTR (green triangles). However, if sorted randomly, (C, where mRNAs are sorted according to the enrichment following miR-15b* transfection) the same tendencies are not seen. Thus, there is specific 3′-UTR enrichment of miR-320 complementary 5′ seeds that correspond specifically to those mRNAs that are most enriched in the Anti-AGO Co-IP and also the mRNAs that are most decreased in the lysate after miR-320 transfection.

Figure 7.

Depiction of some of the novel findings of the current study. (A) miR-320, like other miRNAs that we have evaluated previously (19,22), preferentially recognizes 3′-UTR sequences correlating to the miRNA 5′-seed sequence, and is more likely to induce decreased levels of mRNA targets in the lysates of cells transfected with miR-320. (B) In contrast, miR-107 targets the open reading frame/CDS of mRNAs leading to stable association between AGO and target mRNAs. The main determinant of target binding is the 5′-seed region of miR-107 (red rectangle). However, a miR-107 mutant (MUT-2) with altered 3′ portion (purple rectangle) targets the 3′-UTR sequences. A different miR-107 mutant (MUT-1) with altered 5′-seed portion still targets the CDS, but it targets sequences corresponding to the new 5′-seed portion (yellow rectangle). These data indicate the importance of the 5′-seed sequences in determining the particular mRNAs to target, but also indicate that the 3′ portion of miRNAs may help to select whether the miRNA targets the CDS or 3′-UTR.