mRNA Maturation

exon

sequences retained in mature RNA after splicing

introns

intervening sequences removed from pre-mRNA during splicing

splicing

the process by which introns are excised and exons are joined together to produce mature RNA

introns are also found in some

non-coding RNAs (such as tRNA) and rRNAs in certain organisms

Are all non-coding regions of mRNA introns?

no

exons usually contain coding information, but can also include

untranslated regions

Are the UTR translated into proteins?

no

UTR remain in the

mature mRNA

UTR play a major role in

translation regulation, mRNA stability, localization, and degradation

Are all non-coding sequences removed by splicing?

no

introns are extremely common in

vertebrate nuclear genes

most __ lack introns

most bacterial and archaeal protein-coding genes

many __ contain relatively few introns

unicellular eukaryotes

how many protein-coding genes does the human genome contain

20,000

about how many introns are on each gene

8-9

size of exons

relatively short

size of introns

usually much longer and more variable than exons

exons frequently correspond to

modular protein domains or structural elements

Some human introns exceed __ of nucleotides

hundreds of thousands

many human exons are less than __ nucleotides long

200

most splicosomal introns contain conserved sequence elements, including:

• 5’ splice site

• branch point sequence

• polypyrimidine tract

• 3’ splice site

What do the conserved sequence elements in splicosomal introns do?

guide spliceosome assembly and splicing accuracy

The spliceosome contains

5 major small nuclear RNAs (U1, U2, U4, U5, and U6) and associated proteins

spliceosome function

• recognize splice sites

• bring distant RNA sequences together

• catalyze RNA arrangements

• promote exon ligation

self-splicing intron

introns that can catalyze their own excision without a spliceosome

self-splicing enzymes are

ribozymes

self-splicing enzymes are wide spread in

bacteria, organelles, fungi, and lower eukaryotes

are self-splicing enzymes present in the human nuclear genome

no

first reaction of splicing

2’ hydroxyl group of the branch point adenosine attacks the 5’ splice site, cleaving the exon-intron junction, with the intron forming a lariat intermediate

second reaction of splicing

• The free 3’ hydroxyl group of the upstream exon attacks the 3’ splice site

• the two exons are ligated together

• intron lariat is released and later degraded

Do the reactions of splicing directly require ATP hydrolysis?

no

alternative splicing

a regulated process in which different combinations of exons are joined together from the same pre-mRNA

alternative splicing dramatically expands

proteomic diversity in eukaryotes

different isoforms can possess different

• functions

• localizations

• interaction patterns

• or regulatory properties

What is the result of many exons encoding relatively independent structural or functional domains?

alternative exon combinations can rearrange modular protein elements while still producing folded and functional proteins

the modular organization of exons is a

major evolutionary advantage of exon-intron architecture

protein data bank (PDB)

a pubic international repository containing experimentally determined 3D structures of proteins, nucleic acids, ribonucleoprotein complexes, and large molecular assemblies

At the time of publication, what did the protein data bank’s spliceosome structure represent?

the most complete and highest resolution views of a functional spliceosome intermediate

in structural biology, resolution reflects

the smallest distinguishable

One angstrom (Å) is equivalent to

1 nm or 10^-10m

the lower the angstrom (Å), the __ the resolution and _ the models

The higher the resolution and the more accurate the models

atomic-level detail generally requires resolutions closer to about __ angstroms (Å) or better

2

What can you see at about 3.8 angstroms (Å)?

The overall molecular architecture and many side chains and RNA helices can be assigned

What is not fully resolved at about 3.8 angstroms?

individual atoms

the spliceosome structure was solved using

cryo-electron microscopy (cryo-EM)

cryo-electron microscopy (cryo-EM) is especially powerful for

very large, flexible, and heterogeneous molecular complexes (like the spliceosome)

cryo-electron microscopy (cryo-EM) captured the spliceosome shortly after

the first catalytic step

what did the researchers using cryo-electron microscopy (cryo-EM) do to capture the spliceosome?

• assemble spliceosomes in vitro

• provided only the components necessary for the first catalytic step

• purified complexes corresponding specifically to that stage using biomedical selection strategies (allowing visualization)

Roles of ATPases

• remodel RNA-RNA interactions

• rearrange spliceosomal components

• drive conformational transitions throughout the splicing cycle

Remodeling events by ATPases are essential for

spliceosome assembly, catalysis, proofreading, and disassembly

structural model

snapshots of molecular states

are structures alone sufficient enough to prove molecular mechanisms

no

to validate proposed mechanisms, researchers typically combine structural biology with:

• mutagens

• biochemical assays

• genetics

• functional experiments

Angstrom (Å)

standard scale for atomic and molecular distances

branch site

a conserved sequence within an intron that serves as the attachment point during splicing

cryo-electron microscopy (cryo-EM)

a structural biology technique in which frozen, hydrated samples are imaged with an electron microscope at cryogenic temperatures, and 3D structures are reconstructed from thousands of 2D projections

Lariat

lariat-shaped (loop tail) intermediate formed during pre-mRNA splicing

nuclear magnetic resonance (NMP)

a spectroscopic technique that exploits the magnetic properties of certain atomic nuclei to determine molecular structure

small nuclear RNA (snRNA)

short RNA molecules found in the nucleus, complexed with proteins to form snRNPs

small nuclear ribonucleoprotein particle (snRNP)

a complex of small nuclear RNA molecules bound to a set of specific proteins

structure resolution

degree of detail with which a molecular structure is determined, typically reported in angstroms