second report- eric

8/7/2019 Second Report- Eric

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Examining the miRNAs Involved in HOX Regulation

Travis Babola and Eric Gladstone

Biology Abstract

Micro RNAs, or miRNAs, are short ribonucleotic sequences found in most eukaryotic cells.Before they are processed into their final mature form of approximately 22 base pairs, thesemiRNAs are part of a larger pre-miRNA structure. This structure typically contains one stemloop and one to three internal loops, but can vary. Once this ribonucleotide is processed into itsmature form, it can bind to other mRNAs. Once bound, the resulting dsRNA is broken down,essentially downregulating the resulting gene. This study seeks to examine the secondarystructure of the pre-miRNA with mature forms downregulating HOX genes, which play anessential role in development. Then, we will examine how closely paired the mature miRNA is tothe target gene.

Biology Goals/Methods

Note: Eric and Travis will both be performing all of these steps, but on different miRNAsas stated below.

1. Predict the secondary structure of miRNAs that downregulate HOX gene expression. Asof now, Travis will be examining mir-10 and Eric will be examining mir-181.

a. This secondary structure will be compared to the predicted structure on RFAMusing covariation analysis (Leis Program)

b. Use various resources to add as many sequences as possible to the pool.i. Includes RFAM, Entrez, and, hopefully, RNAmotifii. Alignment using MAFFT

1. Determine how conserved the mature miRNA sequences within the pre-miRNA structureare.

1. Examine how closely paired the mature miRNAs are to the target gene.

Preliminary results on miR-181: Eric

The initial transcript of a miR-181 hox-regulating miRNA must be processed before it isconsidered mature and functional. The RNase enzyme Drosha cuts the initial transcript known as the pri-

miRNA down to a pre-miRNA of roughly 76 nucleotides in length. It is subsequently processed by the

enzyme Dicer to a length of approximately 22 nucleotides.

The mature miR-181 originates near the 5 end of the pre-miRNA. While most of the nucleotidesof the pre-miRNA are not retained in the mature form, they play a critical role in forming the secondary

structure which is required by Drosha for processing. The secondary structure of miR-181 is

characterized by one step loop and three internal loops. Red dots in the figure below indicate nucleotides


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of the pre-miRNA retained in mature miR-181:

Colored nucleotides which are base-paired illustrate the covariance of miR-181 pre-miRNA.

This complementarity between distant nucleotides creates a folding pattern unique to this miRNA. Whileinternal loops decrease the stability of an RNA folding pattern, the majority of miR-181 pre-miRNA

consists of nucleotides with stabilizing canonical base-pairs. Many isoforms of miR-181 display non-canonical base-pairing. This is depicted in the figure below:


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Mutations in this sequence can disrupt proper folding and processing. However multiple alignments

suggest that mutations within loop regions of the pre-miRNA are more tolerated. Below is a MAFFTmultiple alignment of pre-miRNA sequences from RFam:

(FIGURE ISAVAILABLE IN FIRST REPORT)


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The most highly conserved region is near the 5 end of the pre-miRNA. This is expected, as the

mature miR-181 comes from this region. The least conserved region is in the middle of the pre-miRNA,which in comprises the stem loop (nucleotides 34-46). The 3 end is highly conserved as well, to meet

the requirements for proper base pairing in secondary structure formation.In order to analyze these diverse sequences using a covariation program, it is useful to manually

reorganize them by similarity:



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Although these sequences are not identical, they have evolved to retain the same folding pattern.To attain miR-181 sequences by identical secondary structure, RNAMotif was used. At this stage,

developing an accurate descriptor is our priority. The total library of miR-181 sequences attained camefrom Genbank, miRBase, RFam, and BLAST.

NCBI miR-181 nucleotide search results:



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BLAST search results using NCBI sequence titled Musmusculus microRNA 181a-2 (Mir181a-2),


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microRNA:

Running RFam sequences through Leis covariance program provided support for the secondarystructure on RFam. Re-ordering the MAFFT alignment by sequence similarity and running subsets of the

alignment helps get a more accurate covariance ouput.


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One challenge is interpreting the phylogenetic tree for miR-181 sequences because homologous

sequences can in fact be analogous due to convergent evolution. RFam predicts the relationships as

follows:


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In order to evaluate covariance, miR-181 gapped FASTA sequences from RFAM were run

through Leis covariation program (version 1.9).

The output from the mutual information calculator was saved in Excel in order to create a

nucleation file for helix extension.


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The output from helix extension was:

Core pair: nucleotide 52:74 column 78:102

Core pair: nucleotide 52:75 column 78:103Extended: nucleotide 53:74 column 79:102



Extended: nucleotide 52:75 column 78:103



Core pair: nucleotide 54:57 column 80:83Extended: nucleotide 53:58 column 79:84






















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Extended: nucleotide 58:68 column 84:96Extended: nucleotide 59:67 column 85:95




























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Examination of the output indicates that the longest helix in miR-181 is 4 nucleotides long. The

nucleotide pairs described in these helices however do not correspond to those illustrated in the

secondary structure provided on RFAM, and most are far from any pattern seen in any miRNA. These

types of results are duplicated even when different subsets of sequences are put through the

covariation program.

In order to analyze the conserved secondary structure of miR-181, three sequences were

selected to be analyzed manually. The three sequences chosen werefrom P. troglodytes, M. musculus,

and P. paniscus. These sequences were chosen because many other organisms had sequences very

similar to them.


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These three sequences were exported into FASTA format to begin manual examination.

>P.paniscus.2/1-74 AY866165.1/507-580

cuccaag-gaacauucaacgcugucggugaguuugggauuug-------aaaaaaccacugaccguugacuguaccuugggg

>P.troglodytes.10/1-75 AACZ02182759.1/730-804

uuugggg-gaacauucaac-cugucggugaguuugggcagcu-----caggcaaaccaucgaccguugaguggacccugagg

>M.musculus.1/1-76 CAAA01067388.1/1862-1937

cuucagu-gaacauucaacgcugucggugaguuuggaauuc-----aaauaaaaaccaucgaccguug--auuguacccuauag

Lines connecting two nucleotides indicate base pairing in the pre-miRNA. This method of

secondary structure prediction presents many challenges. It is difficult to estimate thermodynamic

stability of one possible structure over another. Non-WC base pairing likely occurs, however was not

used in this method.


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The above figure depicts a plausible structure for P. paniscus miR-181. This structure mostclosely resembles the one presented on RFAM. This possible structure can be visualized better folded.

A rough depiction of this is as follows.


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This overall form resembles that of the structure provided on RFAM.

However, structures for P. troglodytes and M. musculus are slightly less uniform. The number

and location of internal loops varies. This provides a challenge in understanding how Dicer recognizes

such pre-miRNAs. Altering the length or sequence composition of a miRNA can greatly impact its

regulation of its target mRNA. Prior literature reports that miRNA-mRNA interactions display up to 50%

mismatches (Krol 2004).


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Considering tertiary structure adds to the complexity of secondary structure prediction.

Unpaired nucleotides within internal loops or the stem-loop could interact with one another. The

number of possible structures increase exponentially, but thorough covariance analysis provides a

means of identifying potential base pairs.

According to RFAM, miR-181 is preferentially expressed in the B-lymphoid cells of mouse bonemarrow, as well as in the retina and brain. Performing a nucleotide BLAST search for a gene sequence

whose transcript is complementary to the 5 stem of pre-miR-181 may identify other potential targets

for the miRNA besides HOX transcripts.

The next step is to run an RNAmotif descriptor generated for miR-181 to identify additional

sequences from Genbank and to eliminate sequences provided by RFAM which are unrelated.

Currently, Travis and I are creating a Powerpoint which will elaborate on these important

aspects of HoxmiRNAs.

second report- eric

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