Summary 12: RNA Maturation & Processing
Slide 1: We continue our RNA series with RNA processing. The primary focus will be on premRNA modifications at the 5’ and 3’ ends and on splicing, which will convert pre-mRNA into
“mature” mRNA. Importantly, 5’ and 3’ modifications happen only in eukaryotes, whereas
splicing has been observed in both pro- and eukaryotes. However, coding RNAs in bacteria
usually do not require splicing (as shown in this slide), splicing in prokaryotes is confined
to non-coding regulatory RNAs.
Slide 2: This overview puts all processing events into cellular context. We are focusing on
process 1, 2 and 7. Please note that pre-mRNA splicing and polyadenylation occur cotranscriptionally. This slide also informs you about the localization of the three different RNA
polymerase complexes (nucleus vs. nucleolus).
Slide 3: To better understand the differences between transcription in pro- and eukaryotes, we
are taking a step back and look at the gene structure in bacterial DNA. Genes that work in the
same biochemical pathway are often clustered together in an operon. The trp operon
contains five genes that encode proteins required for tryptophan biosynthesis. All genes in this
operon are transcribed from a single promoter into a single mRNA. This mRNA has five
docking sites for ribosomes and thus five different proteins are produced from one mRNA.
Slide 4: In eukaryotes, the five corresponding genes are located in different chromosomal
regions. Each gene gives rise to a transcript that is then translated into protein.
Slide 5: This is an overview of the different pre-mRNA processing steps: 5’ capping, splicing
and 3’ polyadenylation. Splicing occurs during transcription, so strictly speaking, the order of
events is not described quite accurately in this slide. Polyadenylation is triggered by a specific
sequence in the transcript. An endonuclease cleaves this sequence and a specific polymerase
adds a tail of As. This poly (A) tail protects the transcript from 5’ exonucleolytic
degradation and is also required for translation initiation.
Slide 6: The capping enzyme adds a 7-methylguanylate to the 5’ phosphate of the transcript.
This is the only 5’ to 5’ linkage in nucleic acids. The cap protects from degradation and is
important for translation. The capping enzyme is recruited by the phosphorylated CTD of
RNA polymerase II.
Slide 7: This slide illustrates the splicing of intron DNA. The mature mRNA is intronless and
only contains the information encoded in the exons. Typically, there is a short stretch at the 5’
and 3’ ends that is not translated into protein, termed UTR for untranslated region.
Slide 8: Introns are encoded by specific sequences at the exon intron junctions. Each intron has
a 5’ splice site, a 3’ splice site and a branch point. The conserved nucleotides within the
intron are: GU (5’), A (branch point) and AG (3’). The 5’ exon usually ends with an AG
and the 3’ exon starts normally with a G.
Slide 9: We’ve discussed this table before, and I included it as a reminder that intron DNA
amounts to a total of more than 50% of our genome. So the likelihood that a mutation occurs in
an intron is quite high. Some of these mutations can affect the recognition sequences for
splicing, which could cause a pre-mRNA not to be properly processed. The table in slide 19 lists
diseases that arise from improper splicing. We will get back to this point later in the context of
“alternative splicing”.
Slide 10: Introns in human genes are all excised by spliceosome-catalyzed splicing. However,
evolutionarily, splicing likely originated as an RNA-catalyzed process. Group I and II selfsplicing introns utilize a similar splicing mechanism without any requirement for proteins to
catalyze the reaction. Group I introns need a guanosine nucleotide co-factor. Group II employs
the 2’OH of the adenosine at the branch point to “launch” the attack of the 5’ splice site.
This is also true for spliceosome-catalyzed intron removal.
Slide 11: This slide shows how the intron forms a lariat structure. This lariat is eventually
degraded.
Slide 12: Here you can follow the process step-by-step: 2’OH attack of the 5’ splice site,
3’OH is formed after the first transesterification, the 3’OH then attacks the 3’ splice site
followed by a second transesterification. In the end, the two exons are linked and the lariat
intron is excised.
Slide 13: The spliceosome consists of 5 small nuclear ribonucleoprotein particles (snRNPs) all
of which contain a specific small nuclear RNA. The snRNAs provide the specificity, as they
hybridize with the signal sequences of the intron. In vitro, splicing can be carried out in the
absence of proteins. In vivo, the proteins make the process faster and more efficient, but in
the absence of the snRNAs the snRNPs would be useless.
Slides 14 - 17: These slides provide an overview of the step-by-step assembly of the
spliceosome. The individual snRNPs bind specific sequences and bring them close together so
that the transesterifications can occur. For example, U1 and U2 bring the branch point and the 5’
splice site together. Once this has been accomplished, U1 is dispensable. U6 is the catalytic
center, but it is only active once U4 has been removed.
Slide 18: Alternative splicing depicts the process by which a pre-mRNA can yield two
different mature RNAs. This is highly tissue-specific. Alternative splicing creates a lot of
protein variants or isotypes that are not encoded by their own genes. Calcitonin expression
in neuronal and thyroid cells is a well-known example.
Slide 19: Alternative splice variants that produce non-functional or not fully functional proteins
can cause diseases. In most of the cases listed the pre-mRNA is spliced in an alternative incorrect
way, because of mutations in the common splicing signal sequences. Remember BRCA1 or Lesh
Nyhan syndrome from previous lectures?
Slide 20: I hope you can appreciate the complexity of pre-mRNA processing to obtain a mature
and “functional” mRNA. However, not every properly spliced mRNA is necessarily designated
for protein translation. miRNAs are short regulatory sequences that can interfere with the fate
of an mRNA. Most miRNAs target transcripts in their 3’UTRs (slide 7). If base-pairing
between the miRNA and mRNA is discontinuous and incomplete, translation of the
transcript is usually inhibited. If base-pairing is perfect, the target mRNA is degraded or
“silenced”. The class of miRNAs that can trigger silencing through degradation is called
siRNA. RNA interference plays an important role during development, as it can reversibly
or irreversibly suppress specific transcripts.